DETERMINATION OF THE APPARENT KINETIC PARAMIZTERS OF THE NAT2-CATALYZED ACETYLATION OF A SERIES OF

SULFONAMIDE-RYDROXYLAMINES AND SULFONGMaaDES

Catherine Love

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Chemistry University of Toronto

O Copyright Catherine Love 1997

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canada

To my mother

Series of Sdfonamide-hydroxylamines and Sulfonamides

Master of Science 1997

Catherine Love

Graduate Department of Chemistry

University of Toronto

Abstract

The human arylamine N-acetyltransferase (NAT) enzymes catalyze the transfer of

acetate fkom endogenous AcCoA to a variety of substrates including arylarnines and N-

hydroxy arylamines. The su1 fonamide antibio tics are among the substrates whic h undergo

metabolism by NAT and other biotransformation enzymes. The apparent kinetic parameters V,,

and Km were determined for the NAT2-catalyzed acetylation of a senes of sulfonamide-

hydroxylamines and their parent sulfonamides using human recombinant NAT2 enzyme and an

established enzyme assay. NAT2 catalyzed the acetylation of the oxygen of the sulfonamide-

hydroxylamine and the nitrogen of the sulfonaqide substrate. NAT2 displayed a higher affinity

for the hydroxylamine substrates relative to the respective parent sulfonamides. Within the

series of substrates studied, the substrate affinities of NAT2 increased as the nurnber of rnethyl

groups on the pyrimidine ring increased, and as the nurnber of nitrogens in the NI-substituent

decreased. The apparent V,, values varied by less than one order of magnitude for al1

substrates. Both the apparent V,, values and the specificity constants (Vmax/Km) were found to

increase with the number of methyl groups on the pyrimidine ring. Previous research has

illustrated a similar trend in the rates of acetylation by NAT2 in vivo.

iii

To Dr. McClelland for your guidance and support throughout rny research and studies.

To Dr. MacMillan for reading my thesis.

To my labmates- Pratima, Eddy, Daniel, Thuy, Abid, Cristina and Patrick- for your encouragement and fiiendship.

To Geoff for your work, advice and coffee.

To my al1 of my fkiends and colleagues in the Department of Chemistry, especially Steve and Dom for the key, Pat for walking the halls with me, and Grace for Pilates.

To the blondies- Jenn, Natalie, Kendra, Amanda, Angela and Kerri- and Blake and Ailan for listening when you had no idea what 1 was t a h g about.

To Bridget, John and Bernie for al1 of your love and support.

Abstract .................................................................................................... iii

Acknowfedgments ......................................................................................... iv

Table of Contents ........................................................................................... v

List of Tables ................................................................................................. vi

List of Figures ............................................................................................. vii

... List of Appendices ........................................................................................ viii

Abbreviations ................................................................................................ iv

Introduction ..................................................................................................... 1

Materials and Methods ............................................................................... 20

............................................................................................................ Results 26

Discussion ..................................................................................................... 50

References ..................................................................................................... 64

Table 1 : Incubation of SMZ-NHOAc in phosphate and NAT assay buffer with .................................................................................. and without DTT 28

Table 2: Protein concentrations of lysate batches used for .................................................... NAT2-sulfonamide-hydroxylamine assays 31

Table 3: HPLC conditions and retention times used for .................................................... NAT2-sulfonamide-hydroxylamine assays 32

Table 4: Approximate impurity contents of SULF-NHOH substrates ..................... 34

Table 5: Approximate impurity contents of SULF-NHOAc product standards .......... 35

........................................... Table 6: Results of the NAT2-SMZ-NHOH assays 37

........................................... Table 7: Resdts of the NAT2-SMR-NHOH assays 38

........................................... Table 8: Results of the NAT2-SDZ-NHûH assays 39

............................................ Table 9: Results of the NAT2-SPY-NHOH assays 40

.......................................... Table 10: Resdts of the NAT2-SAA-NHOH assays 41

........................... Table 1 1 : Reaction conditions for the NAT2-sulfonarnide assays 44

.................................................... Table 12: Results of the NAT2-SMZ assays 45

................................................... Table 13: Results of the NAT2-SMR assays 46

.................................................... Table 14: Results of the NA=-SDZ assays 47

.................................................... Table 15: Results of the NAT2-SPY assays 48

..................................................... Table 16: Results of the NATZSAA assay 49

Table 17: Apparent khetic parameters for the NAT2-catalyzed acetylation of .................................................................. Series 1 and Series 2 substrates 51

Table 18: Cornparison of the elimination half lifes to the apparent specificity ........................................................ constants for SMZ. SPY. SDZ and SM R. 57

.............................................................................. Figure 1: Isoniazid 4

....................................................................... Figure 2: SMZ and PAS 5

...................................................... Figure 3: Reactions catalyzed by NAT 6

Figure 4: The proposed catalytic mechanism of AcCoA: N-hydroxyarylamirie O-acetyltrans ferase of Salmonella typhimur ium .................... 8

Figure 5: Graph of v vs . [SI for an enzyme displaying Michaelis-Menton ............................................................................................ kinetics 9

................ Figure 6: Mechanism of the Acetyl Coenzyme A regenerating system 11

.................................. Figure 7: Potential pathways of arylamuie rnetabolism 12

................................................................................ Figure 8: PABA 14

............................................................ Figure 9: Sulfonamide structures 15

... Figure 10: Suifonamide metabolism and possible fornation of toxic metabolites 17

Figure 1 1 : Rate of NAT2-catalyzed conversion of SMZ-NHOH to ......................................... SMZ-NHOAc at incubation times 2 to 60 minutes 27

Figure 12: Possible mechanisms for the conversion of SMZ-NHOAc to ......................................................................... SM and SMZ44HOI-I 30

................................................... Figure 13: Sample HPLC chromatogram 33

...................... Figure 14: Sample reaction rate vs . substrate concentration curve 36

Figure 15: Ratio of apparent Km values of SULF-NHOH and SULF substrates ......................................................................... in Series 1 and Series 2 52

................................... Figure 16: Apparent Km values for Series 1 substrates 53

.................................... Figure 17: Apparent K,,, values for Series 2 substrates 53

.................................. Figure 18: Apparent Vmm values for Series 1 substrates 54

.................................. Figure 19: Apparent Vmm values for Series 2 substrates 55

............. Figure 20: Rate vs . substrate concentration curves for Series 1 substrates 56

............. Figure 2 1 : Rate vs . substrate concentration curves for Series 2 substrates 56

vii

.............................. Appendix A: Synthesis and Characterization of Sulfonamide Derivatives 67

Appendix B: Preparation of the E . coli NAT Lysate .............................................................. 75

..................... Appendix C: Data and Rate vs . Substrate Concentration Curves for Al1 Assays 76

AcCoA - acetyl CoA

AUFS - Absorbance Units Fu11 Scaie

AUhmol - Absorbance Units/nmole (unit for sensitivity factors)

BSA - bovine serum albumin

CYP45O - cytochrome P450

DMSO - dimethyl sulfoxide

DNA - deoxyribonucleic acid

DTT - dithiothreitol

EDTA - ethylenediarninetetraacetic acid

HPLC - High Performance Liquid Chromatography

kat - the catalytic rate constant

Km - the Michaelis constant

v - enzyme velocity

NADPH - nicotinamide adenine dinucleotide

NAT - arylamine N-acetyltransferase (1 OR 2)

PABA - p-arninobenzoic acid

PAS - p-aminosalicylic acid

SAA - sul fanilanilide (4-amino-N-2-phenyl benzenesul fonamide)

SDZ - sulfadiazene (4-amino-N-2-pyrimidinylbenzenesulfonamide)

SMR - sulfamerazine (4-amino-N-(4-methyl-2-pyrimidinyl)benzenesulfonamide)

SMZ - sulfarnethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)bemenesdfon~de)

SPE - sulfaperine (4-amino-N-(5-methyl-2-pyrimidinyI)benzenesulfonamide)

SPY - su1 fapyridine (4-amino-N-2-pyridiny lbenzenesul fonamide)

SULF-N(0H)Ac - the hydroxamic acid derivative (N-acetylated sulfonmide-hydroxylamine) of the sulfonamide

SULF-NHOAc - the acetoxy ester derivative (O-acetylated sulfonamide-hydroxylamine) of the sulfonamide

SULF-NHOH - the N1-hydroxylamine derivative of the sulfonamide

- elimination half life

UDP - uridine diphosphate

V,,, - the maximal velocity of the enzyme

Overview of Xenobiotic Transformation

Humans and other organisms are regularly exposed to a wide range of foreign

compounds known as xenobiotics. These chernicals are absorbed across the skin or lungs,

or may be ingested in food and drink, or in the f o m of pharmaceuticals. Many xenobiotics

are highly lipophilic, and the human body utilizes a variety of enzyme systems which

function to convert these lipophilic compounds to more polar, water-soluble products that

may be more readily excreted. While this biotransfonnation may decrease or eliminate any

biological activity which the parent xenobiotic possessed, the metabolites rnay themselves

be active. Thus, overall the effect may enhance the activity or toxicity of the compound.

The enzyme reactions which serve to biotransform xenobiotics are generally divided

into two classes, namely Phase 1 and Phase II reactions. Phase 1 metabolism involves the

production or unmasking of a functional group such as -OH, -SH or -NH in the cornPound.'

UsualIy, the first step toward detoxification and elimination at this stage is the introduction

of oxygen into a compound? The cytochrome P-450 (CYP450) enzymes perform the

majority of these Phase 1 reactions, including C- and N-hydroxylation. These enzymes

catalyze the insertion of one atom of molecular oxygen into a substrate, while the other

oxygen atom is converted to water using electrons which are donated by NADPH via

NADPH cytochrome P-450 reductase. CYP450s are found primarily in the liver, and more

than twenty forms have been identified and characterized in human tissues.) Phase II

rnetabolism reactions conjugate highly hydrophilic moieties to polar groups such as the ones

introduced by Phase 1 reactions. Endogenous substrates such as glutathione, UDP-

glucuronic acid, phosphoadenosyl phosphosulfate or acetyl CoA are utilized by enzymes

such as glutathione transferase, UDP-glucuronyltransferase, sulfotransferases or N-

acetyltransferase respectively to supply the necessary chemical groups for ~on ju~a t ion .~

xenobiotics fkom the body. Unfortunately, the result of these biotransformations may also

activate compounds to electrophilic foms with increased toxicity and carcinogenicity.

Evolution of Metaboiic Enzymes

Xenobiotic biotransfomation enzyme systems have evolved in several ways to cope

with chemicals which are foreign to the body. There may exist many closely related foms

of an enzyme which have distinct or overlapping substrate specificities, intended to cope

with many divergent chemical structures. Individual enzymes also appear to have evolved

to metabolize a wide range of xenobiotics. This contrasts with enzymes involved in

biosynthetic pathways which generally display rigid substrate specificity. Indeed, there are

approximately 200 000 xenobiotic substrates for the cytochrome P450 enzymes.5 In some

cases, this enhanced substrate acceptance allows a single enzyme to biotransfonn hundreds

of different chemicals in an effort to ensure complete biotransformation of al1 exogenous

~ o m ~ o u n d s . ~

Some enzymes metabolize both endogenous and exogenous substrates, but appear

to show a preference for one or the other. For example, of the ten families of cytochrorne P-

450 genes which exist in mamrnalian species, some families are responsible for the

metabolism of endogenous compounds such as steroids and lipids, while three farnilies,

namely CYP1, 2, and 3 prirnarily metabolize xenobiotics. These are the hepatic mixed-

function oxidases, which may be said to have evolved in response to environmental

stresses.' Metabolism of endogenous substrates is also accomplished by some of the Phase

II enzymes, such as catechol O-methyltransferase. However, there are some enzymes, such

as arylamine N-acetyltransferase, for which there are presently no known endogenous

substrates. This suggests that some enzymes have evolved solely in an effort to protect an

organism fiom environmental xenobiotic~.~

- --- --- 7 ---- ------- -- ---r-- -Y"--. YI--*--

Individuals within a population may have varied responses to drugs and other

substances that undergo biotransformation. Many factors may influence the difference in an

individual response to a xenobiotic, including diet, state of health and exposure to other

xenobiotics. Genetic variants of enzymes of biotransformation can also cause these

d i f f e ~ g responses. These pharmacogenetic differences are often the result of monogenic

and polymorphic genetic variants. That is, the variation occurs at one gene locus to produce

phenotypes that are distinguishable to the extent which the least cornmon occurring

phenotype has a frequency of geater than 1% in the tested population.8 These

polymorphisms can result in the subdivision of human populations into 'poor' and 'extensive'

metabolizers. As an example, differences in aromatic amine metabolism in humans is

thought to exist due to the polymorphism of the enzyme arylamine N-acetyltransferase 2.

There rnay also be polymorphisms in other metabolic enzymes such as cytochrome P450

1A2 and the thermostable sulfotran~ferase.~

Arylamine N-Acetvltransferase and the Human Acetvlation Polymorphism

One of the best studied examples of a genetic polymorphism of a xenobiotic-

metabolizing enzyme is that of arylamine N-acetyltransferase (NAT; EC 2.3.1.5). In the

1950's, therapy with the antitubercular drug isoniazid (Figure 1) resulted in a significant

incidence of unwanted side effects.I0 Population studies illustrated a bimodal frequency

distribution which enabled the segregation of individuals into 'rapid' and 'slow' eliminators

of the drug from the body1'. Studies of elhination patterns in family pedigrees and twins,

and interethnic differences in the proportions of slow and rapid eliminators, combined to

identify the genetic nature of this variation in the metabolism of isoniazid." The N-

acetylation of isoniazid primarily by a hepatic enzyme was found to be the cause of these

population variations. This reaction is now known to be catalyzed by the Phase II cytosolic

enzyme NAT. In the case of isoniazid, the adverse affects due to treatment with the drug

WGIG LUUU 111 irloiviauais wui me slow aceryiaror pnenotype. Unacetylated isoniazid could

not be effectively eliminated form the body, and the adverse affects followed. Individual

differences in the metabolism of xenobiotics, which may be dmgs used in medical therapy

or prevention, chemical carcinogens in the workplace or elsewhere in the environment, or

the products of Phase 1 metabolism are attributable in part to the polymorphism in the

acetylator genes.

Figure 1 : Isoniazid

NAT exists as two genes, namely NAT1 and NAT2. The NATl gene encodes the

NAT1 protein, which consists of a single polypeptide chah of 290 amino acids that is likely

expressed in most t i s s~es . ' ~ The NAT2 enzyme is the product of the NAT2 gene, and is a

protein of the same size that is expressed tissue-selectively in liver hepatocytes and

duodenal mucosa.l3 NAT2 is the site of the human acetylation polymorphism, which

divides individuals into rapid and slow acetylators. Rapid acetylators are either

homozygous or heterozygous for the wild-type NAT2 alleles. Slow acetylators possess

variant NAT2 alleles, resulting in decreased levels of NAT2 activity in human b e r cytosol

due to poor expression, instability or decreased catalytic activity. As a result, the overall

metabolism of many exogenous compounds is affected by this genetic polymorphism.

Although NAT 1 and NAT2 share 8 1 % amino acid sequence homology, they

exhibit distinct kinetic characteristics." Thus NATl possesses a high degree of selectivity

for para-aminosalicylic acid (PAS), while NAT2 prefers and sulfamethazine (SMZ). (Figure

2) Regardless of acetylator phenotype, the rate of acetylation of NAT 1-selective substrates,

also known as monomorphic substrates, is relatively unaffected. Whereas acetylation rates

of polymorphic NAT2-selective substrates depends upon acetylator phenotype.

Figure 2 : SM2 and PAS

Sulfamethazine (SMZ) p-aminosalicylic acid (PAS)

NAT-catalvzed Reactions

NAT enzymes are capable of conjugating acetate to a variety of substrates. It has

been either confirmed or suggested that NAT catalyses the following reactions: 1) N-

acetylation of primary aromatic amines, 2) N-acetylation of hydroxylamines, 3) 0-

acetylation of hydroxylamines, 4) O-acetylation of arylhydroxamic acids, 5) N-acetylation

of arylhyroxylamine O-acetates, 6) intramolecular N,O acyltransfer of arylhydroxamic

acids, 7) N-acetylation of prulzary aromatic hydrazines, and 8) N-acetylation of primary

heterocyclic (Figure 3). Intrarnolecular N,O acyltransfer is the only reaction for

which AcCoA is not required.

The NAT-catalyzed reactions listed above require that the nitrogen or oxygen atom

to which acetate is conjugated be directly attached to, or not more than one intervening

nitrogen atom away fiom, the homo- or heterocyclic aromatic ring. It has been

demonstrated that NAT does not readily accept aliphatic and arylakylamine substrates.I6

This may be a result of the NAT enzyme protein structure, which has yet to be elucidated.

1 ) N-acetylation of prhary arornatic amines

2) N-acetylation of primary aromatic hydroxylamines - OH

3) O-acetylation of primary aromatic hydroxylamines

4) O-acetylation of aryl hydroxamic acids

7) N-acetylation of primary aromatic hydrazines

8) N-acetylation of primary heterocyclic amines

NAT catalyses the transfer of acetate fi-om acetyl Coenzyme A (AcCoA) to a

suitable substrate via a ping-pong bi-bi reaction mechanism." This mechanism is

characterized by a two-stage reaction mechanism where a functional group of the first

substrate is displaced fiom the substrate by the enzyme to yield a product and a stable

enzyme intermediate. Next, a second substrate displaces the enzyme-bound functional

group to yield the second product." In the case of NAT, the reaction that occurs is the

following :

NAT + AcCoA - NAT-Ac + CoASH (1)

NAT-Ac + Ar-NH2 - NAT + Ar-NHAc (2)

In the first step of the reaction, (l), the acetyl group (-COCH3 (Ac)) of AcCoA is

transferred to the enzyme to produce an acetyl-enzyme intermediate and Coenzyme A. The

second reaction transfers the acetyl group fiom NAT to a suitable substrate such as an

arylamine (Ar-NH2). This regenerates the enzyme and produces the acetylated arylamine

( A r - W C ) product.

The NAT Active Site

Although the protein structure of NAT remains unknown, a catalytic mechanism of

the enzyme has been proposed. In a study conducted by Watanabe and cow~rkers '~ the

nucleotide and corresponding amino acid sequences in O-acetyltransferase of Salmonella

typhimurium and arylamine N-acetyltrans ferases of human, rabbit, hamster and chicken

origins were compared. The resuits revealed a highly conserved region of hg-Gly-Gly-X-

~ ~ ~ 6 9 . The cysteine at position 69 of S. typhimurium was conserved in the sarne sequence

at position 68 of the higher organisms. A mutant O-acetyltransferase of S. typhirnurium

which contained Ala at position 69 did not show any of the activities related to O and N-

acetyltransferase. Based on this evidence, a catalytic mechanism for the acetyl Coenzyme

A:N-hydroxyarylamine O-acety I tram ferase of S. typhimurium was proposed. (Figure 4)

(fiom Watanabe et al. J. Biol. Chem. 267,8429-8436 (1992))

A study by Dupret and Grant provided aidence for the direct involvement of cys68

in the catalytic mechanism of human NAT^.^' Mutating this residue for Gly resulted in

NAT2 which possessed no enzyme activity. Mutations of other cysteine residues in the

protein produced NAT2 proteins which still possessed some catalytic activity, suggesting

that these were not essentiai for catalysis by NAT2 but were involved in maintaining the

tertiary structure of the protein.

Enzyme Kinetics Studies and NAT

The kinetics of both NAT1 and NAT2 have been studied using a variety of

substrates. The study of enzyme kinetics in general involves the use of the Michaelis-

Menton equation (Equation 1). In order for an enzyme reaction to obey Michaelis-Menton

kinetics, the rate which is measured must be an initial rate, v , for the formation of the first

10-15% of the products where changes in reagent concentrations are usually linear with

tirne. This also assumes that the concentration of the enzyme is negligible compared to that

of the substrate. The initial rate is generally directly proportional to the enzyme

concentration El,. The rate v also follows saturation kinetics with respect to the substrate

k w u k r u u a t . a w u LUJ, JULU U L ~ L QL VLLY IVW 101 , v U~LLGQJGJ i a a l ~ c u i j wluï LOJ, a ~ u u~~ïï UCg1113 L u

increase less rapidly untiI at a saturating concentration, v approaches a limiting value called

V,,. The rate equation which describes this behaviour is the Michaelis-Menton equation.

Equation 1: The Michaelis-Menton equation

The limiting V,, is equal to k, [El, where ha, is called the catalytic constant or turnover

number. The quantity b, represents the maximum number of substrate molecules which

can be converted to products per active site on the enzyme per unit time. The Michaelis

constant, Km, is the substrate concentration which is required to achieve V,,/2. A plot of

reaction rate v vs. substrate concentration [SI for an enzyme-catalyzed reaction which obeys

Michaelis-Menton kinetics would produce a graph such Figure 5.

Figure 5 : Graph of v vu. ($1 for an enzyme displaying Michaelis-Menton kinetics

(From Ferscht, A. Enzyme Structure and Mechanism. W. H. Freeman and Company, (1985)- Second Edition.)

- -- - - . ----------------- v . - * w * - :'J a,,, - *.'.aiL"J',L'J, A'",, W.- m..' . W.

high [SI, v = V,,. At low substrate concentrations kJK, is the apparent second order

rate constant of the enzymatic reaction. The ratio &,,/Km is termed the specificity constant

because it determines the specificity for competing substrates. In cases where the exact

actual enzyme concentration in a reaction is not known, the Vm,/K, ratio rnay be used as a

reasonable estimate of the specificity constant (kJK,).

In most multi-substrate enzyme systems such as NAT, the reactions obey Michaelis-

Menton kinetics when the concentration of one substrate is held constant while the other is

varied. Enzymes which employ ping-pong kinetics yield parallel double-reciprocal plots

when the concentration of one substrate is varied while the concentration of the other is

f i ~ e d . ~ ' Such plots have been demonstrated for NAT-catalyzed acety~ation.'~

In order to study the kinetics of enzymes which display ping-pong kinetics, it is

necessary to maintain the concentration of one substrate at a constant level while varying

the concentration of the other substrate. The results achieved are referred to as "apparent"

values. in the case of NAT, physiologically relevant kinetic parameters may be attained by

maintainhg the level of AcCoA at a constant level of 100 pM which is estimated to be that

found in liver cytoplasrn." This is accomplished with the use of a AcCoA-regenerating

system which uses caniitine acetyltransferase to transfer the acetyl group fiom acetyl D,L-

camitine to CoA, regenerating AcCoA '4 (Figure 6). Under these conditions where the

cofactor supply is fixed, enzyme kinetic parameters may be derived from non-linear

regression of enzyme activity data generated by measuring enzyme velocity v at varying

substrate levels using the previously described Michaelis-Menton equation.

1 (cH&N+-ch-CH-ci+-coo-

Acetyl carnitine

Carnitine Acetyltransferase

(C~~&N+-CH~-~H-C~+-COO- Carnitine

The Relationship of NAT to Carcino~enesis and Adverse Drup Reactions

The metabolism of many arylamine xenobiotics by NAT have been studied

extensively in relation to a wide variety of human disorders including various cancers and

adverse drug reactions. Although it is difficult to determine the route of biotransformation

of a given xenobiotic by the many metabolic pathways available in the body, several

enzymes which compete or combine with NAT-catalyzed acetylation of arylamine

substrates appear to be relevant in these areas of toxicology. Some of the possible

metabolic pathways available to arylamine compounds are illustrated in Figure 7.

rrgurr: 1 ; rutenriai parnways ui aryiamine meraouiism

UDPGT ,H NAT Ac - Ar-N, -

H "--CH H

CYP450

NAT

O a u c UDPGT

NAT

,OAc Ar-N,

+ + A r - L H Ar -N-Ac

Arylnitrenium ions

NAT = Arylarnine N-acetyltransferase UDPGT = UDP-glucuronyltransferase CYP450 = Cytochrome P450 ST = Sulfotransferase

The N-acetylation of arylamines is generally accepted as a detoxification pathway.

Activation of arylamine substrates via oxidation by the cytochrome P450 enzymes such as

CYPIA2 results in the production of the more reactive arylhydroxylamine species. This

arylhydroxylamine has been postulated to be responsible in part for the adverse reactions

associated with sulfonamide antibiotic therapy.25*2"" Subsequent N-acetylation by NAT to

produce the hydroxamic acid is commonly recognized as a detoxiQing pathway for

arylamines. O-acetylation, on the other hand, produces the highly reactive and electrophilic

acetoxy ester species. Decomposition of the N-acetoxy compound c m produce an

arylnitxenium ion which may bind to DNA. Studies of carcinogenic arylamines have

-- -- - - - - - - - - - - -- - - - - - - - - - - --d - - - -4 - ir

critical to the covalent binding of such compounds to DNA.14

Variations in metabolizer phenotype have been found to be related to many of these

issues. Exposure to environmental carcinogens such as 4-aminobiphenyl, 2-naphthylamine

and benzidine results in metabolism via oxidation by CYPlA2 as well as acetylation by

NAT.^*'* slow acetylator status combined with such exposure has been shown to be a

predisposing factor for bladder cancer.2g It has been suggested that the unacetylated

hydroxylamines are produced in the liver, and then enter the circulation, eventually reaching

the bladder where they may be M e r metabolized by NAT1 to the N-acetoxy metabolite

with subsequent formation of a nitrenium ion which binds to urothelial DNA." This

suggests that NAT2 acetylation in the liver may then be a detoxification mechanism. Rapid

acetylator status has been linked to higher incidences of colorectal cancer, suggesting that

the relevant carcinogenic species is the N-acetoxyarylamine produced by NAT2, and

demonstrating that the acetylation polymorphism affects both the metabolic activation and

deactivation of arylamine ~arc ino~ens .~ '

Acetylator phenotype is also associated with adverse h g reactions. The

metabolism of many drugs including isoniazid, many sulfonamides, hydralazine, and

phenelzine are affected by acetylator stat~s.~' In the case of isoniazid, as previously

described, slow acetylators have a higher incidence of adverse reactions than do rapid

metabolizers.

The Metabolism of Sulfonamide Chemotherapeutics

Sulfonamide antibiotics were introduced in 1936.'~ Approximately 150 different

sulfonamides have been marketed since then, and they continue to be among the most

widely used antibacterial agents in the world today.' They are structural analogues of p-

aminobenzoic acid (PAB A) (Figure 8), an essential extracellular requirement for the

formation of folic acid by microorganisms. Purine synthesis requires folic acid, and

V Y - prevents further bacterial growth." In addition to their clinical utility, sulfonamides are also

responsible for a wide variety of adverse h g reactions. These include fever and skin rash,

along with hepatic, renal, bone oarrow, pulmonary, CNS or cardiac toxicity.'' In general,

hypersensitivity or idiosyncratic reactions are usually caused by genetic differences in

metabolism or an immunological response.' In the case of sulfonamides, these reactions

appear to occur because of differences in the metabolism and detoxification of reactive

metabolites of the s~lfonamides~~ or hapten fonnation resulting in an allergic reaction.

Figure 8 : PABA

The overview of metabolism of aryIamines discussed previously may be applied to

the sulfonamides. Sulfonamides are metabolized to a varying extent in tissues, prirnarily in

the l i ~ e r . ~ ~ SdfmethaPne (SMZ), sulfmerazine (SMR), sulfadiazene (SDZ), sulfaperine

(SPE), sulfapyridine (SPY) and sulfamethoxazole (SMX) (Figure 9) are among the

sulfonamides which may be glucuronidated, sulfated, oxidized, or acetylated by the various

metabolic enzymes in the body to form different metabolites.

Sul fame thazine Sulfadiazene

Sulfapyridine

Sulfamethoxazole

CYP45O-catalyzed oxidation of the primary amine nitrogen atom have been postulated to

play an important role in a variety of sulfonarnide adverse reacti~ns?'~~~*" Sulfadiazene and

sulfamethoxazole have both been shown to undergo oxidation by a CYP450 enzyme to the

hydroxylamine in in vitro systems using human lymphocytes and liver microsornes

r e ~ ~ e c t i v e l ~ ? . ~ ~ The toxicity of these hydroxylamine metabolites has also been measured

using synthetic sulfadiazene-hydroxylamine (SDZ-WOH) and sulfamethoxazole-

hydroxylamine (SMX-NHOH) as well as the parent compounds. It was demonstrated that it

is indeed the hydroxylamine derivatives, and not the parent compounds, which were

cytotoxic in vitro to human lymphocytes.'6 The hydroxylamine derivative of SMX has also

been identified as an in vivo metabolite. SMX-NHOH was found to contribute to the total

SMX excreted in a 24 hour penod by healthy volunteers, demonstrating that it is an

authentic in vivo metabolite in h ~ r n a n s . ~ ~

Slow acetylator phenotype has been postulated to be a risk factor for the

development of sulfonamide hypersensitivity reacti0ns.1~ This rnay suggest that it is the

sulfonamide-hydroxylamine which is responsible for mediating these reactions due to the

lack of N-acetylation by NATS. It is also possible however, that the slow acetylator

phenotype makes the dmg available for prior oxidation to the hydroxylamine, and it is this

compound which rnay be subsequently acetylated by slow metabolizers. This would

produce the N-acetoxy species of the sulfonamide which may, like some carcinogenic

arylarnines, mediate the toxicity.

The production of sulfonamide-N-acetoxy derivatives by NAT has been

investigated. In one study, the human recombinant NAT1 and NAT2 enzymes were found

to be capable of converting SMX-NHOH to SMX-N-acetoxy (SMX-NHOAc). It was also

SMX-NHOH, and that SMX-NHOAc appears to undergo metabolism back to SMX-NHOH

and SMX by human peripheral blood mononuclear ce11 fractions." Identification of the N-

acetoxy metabolite in vivo may be difficult due to the instability of this species due to the

acetate ion leaving group and the catabolism back to SMX and SMX-NHOH.

An overview of suifonamide metabolism which may mediate adverse reactions rnay

include the pathways illustrated in Figure 10. The metabolism of the parent compound ro

the hydroxylamine rnay mediate the response. Subsequent acetylation to the N-acetoxy

metabolite may also be responsible for the toxicity of sulfonamides.

Figure 10 : Sulfonamide metabolism and possible formation of toxic metabolites

NAT

H2N - Acetylated Non-toxic Metabolite

Sulfonarnide

NAT

r

N-acetoxy (Reactive Metabolite?)

I Reactive Metabolite 9

w Covalent Binding

Detoxification Pathway

* Non-toxic Metabolite Cellular Toxicity

Irnmunological Response

Clinical Expression

a \ F a a u u u a u a p a YGCWFGU LVIVBG-UI~I U C L U . ~ L U L G auu n . i c . L y L a u u u UR UUIIUU~CIUIUG.S

In an effort to determine the effect of molecular structure on the acetylation of

sulfonamides in humans, several in vivo studies have been conducted. In the series of al1

sulfonamides, it seems that only the 2-sulfanilamido-pyrinzidines and -pyridine(s) are

susceptible to bimodal a~et~lat ion."~ In general, it has been found that the Ni-substituent

modifies the acetylation at the Nd-atom. It has been demonstrated that in the series of 2-

sulfanilamido-pyrimidines consisting of sulfadiazene, sulfamerazine and sulfamethazine, the

absolute rate of acetyIation in both fast and slow acetylators hcreases with increasing

methyl group substitution in the NI-pyrimidine ring?' Al1 three sulfonamides are

considered polymorphic substrates, and increasing methyl substitution also makes the

differences between fast and slow acetylators more visible."

Rationale and Obiectives for this Studv

The goal of the present study is to investigate the NAT2-catalyzed acetylation of the

hydroxylamine derivatives of the sulfonamides SMZ, SMR, SDZ, SPE, SPY and SAA as

well as the acetylation of the parent compounds. It has been demonstrated that the number

of methyl group substitutions in the pyrimidine ring affects the rate at which NAT2

acetylates SMZ, SMR and SDZ in vivo. We will investigate the effects on the apparent

kinetic parameters V,, and Km of modi@ing the Nd-substituent fiom a primary amine to a

hydroxylamine group, the effect of methyl substituents in the NI-pyrimidine ring, as well as

the effect of replacing the pyrimidine with a pyridine or benzene ring. Using recombinant

wild-type NAT2 enzyme and an established enzyme assay, acetylation rates at varying

substrate concentrations of both the sulfonamide-hydroxylarnines and sulfonamides will be

used to construct curves fiom which the values of V,, and Km can be determined. The

apparent substrate specificities will also be examined. We hypothesize that the rates of

acetylation by NAT2 for the previously studied substrates SMZ, SMR, and SDZ will display

--- --- r------- W., Y---- -.- - . . W . -..w W*LWWC Y* -1- 1 L . V I W W U I U I Y U . . I C U . - V - ..LI Y.*."I

,suIfonamides on the kinetic parameters which will be measured for the NAT2-catalyzed

acetylation reactions is not known.

Substrstes and Product Standards

Sdfamethazine (SMZ), sulfapyridine (SPY) and the sodium salts of sulfamerazine

(SMR) and sulfadiazene (SDZ) were obtained fiom Sigma Chemical Company (St. Louis

MO). SDZ was obtained fiom Aldrich Chemical Company (Milwaukee WI). Sulfaperine

(SPE) and sulfanilanilide (SAA) were synthesized and characterized by Dr. Abid Ahmad.

N-acetyl sulfamethazine (SMZ-NHAc) was obtained fkom Dr. Grant's lab, and was

previously synthesized and characterized by Dr. Grant. N-acetyl sulfapyridine (SPY-NHAc),

sulfamerazine (SMR-NHAc), sulfadiazene (SDZ-IWAc) and sulfaperine (SPE-NHAc) were

synthesized and characterized by Dr. Ahmad. The hydroxylamines of sulfamethazine

(SMZ-NHOH), sulfapyridine (SPY-NHOH), sulfamerazine (SMR-NHOH), sulfadiazene

(SDZ-NHOH), sulfaperine (SPE-NHOH) and sulfaniianilide (SAA-NHOH) were also

synthesized by Dr. Ahmad, as were the corresponding N-acetoxy derivatives (SULF-

NHûAc). Dr. Ahmad's syntheses and characterizations are detailed in Appendix A.

The NAT Enzyme System

The E. coli NAT lysates were prepared as described in Appendix B by Geoff

Goodfellow in Dr. Grant's lab at the Hospital for Sick Children (Toronto, ON). The

components of the AcCoA-regenerating system, namely acetyl-DL-camitine, carnitine

acetyltransferase (E.C. 2.3.1.7) and acetyl Coenzyme A (AcCoA) were obtained fiom

Sigma Chemical Company (St. Louis, MO). Dithiothreitol (DTT) for the TEDK and NAT

assay buffers was obtained from Fisher Biotech (Fairhaven, NJ).

Ethylenediaminetetraacetic acid (EDTA), triethanolamine-HC1 and potassium chloride for

Sigma Chemical Company.

The Bio-Rad Protein Assay

Bovine semm albumin (BSA) was obtained fiom Boehringer Mannheim (Germany)

and the Bio-Rad Protein Assay dye binding solution was fkom Bio-Rad (Hercules, CA). UV

spectroscopy was performed on a Carey 2200 W-Vis Spectrophotometer (Varian

Instruments).

Hiph Performance Liquid Chromato~raphy

HPLC of NAT2 assays which utilized sulfonamide-hydroxylamine substrates was

performed using a Waters 600 E system controller, Waters 486 Tunable Absorbance

Detector, Waters U6K Universal Liquid Chromatograph hjector, and a reverse phase Crs

pondpack column (8 mm x 10 cm). Chromatograrns were processed by a Waters 746 Data

Module.

HPLC of NAT2 assays which utilized sulfonamide substrates was performed in Dr.

Grant's lab at the Hospital for Sick Children. HPLC was conducted using an automated

isocratic system (Shimadm Scientific Instrtunents Inc., Columbia MD) which consisted of a

LPI-6B System Controller, a SIL-6B auto injecter, a LC600 piimp, a SPD-6A UV detector

and a reverse phase Cis Beckman Ultrasphere column (4.6mm x 15 cm). The software

program EZChromTM (Scientific Software Inc., San Ramon CA) controlled this system

through an IBM PSI1 cornputer.

High performance liquid chromatography (HPLC) mobile phases consisted of

HPLC grade acetonitrile from Caledon (Georgetown ON), and a buffer made from

deionized water and perchloric acid and sodium perchlorate bom Caledon.

The data generated fiom the HPLC results was analyzed using the ~ r a f i t @ software

program (Erithacus Software Ltd 1 Microsoft Corp.).

The Bio-Rad Protein Assav

The dye-binding method o. '~radford~~ was used to measure the amount of protein in

the individual NAT2 enzyme lysate batches. 100 pl of 0.2 to 1 .O mg/ml BSA Ln TEDK were

reacted with 5 ml of diluted Bio-Rad protein assay solution, assayed for absorbance at 595

nm and used to constnict a standard curve. The absorbance of the lysate was expected to

correspond to approximately Img/ml. Several dilutions of NAT2 lysate were made

accordingly in TEDK b d e r and the absorbance was rneasured at 595 nm. The resultant

protein concentration was used to express the rate of acetylation in moles of acetylated

product per minute of incubation t h e per milligram of protein in the reaction mixture.

The NAT2 Enzvme Assav

The method used for the NAT2 enzyme assay is based on that described by Grant et

al." Final substrate concentrations for the sulfonamide-hydroxylamines ranged fi-om 30 to

1000 ph4 and fuial concentrations of the sulfonamide substrates ranged fiom 30 to 6000

pM. Substrates of ten-fold higher concentration were prepared in 25% DMSO and were

diluted in the reaction mixture by ten times to achieve a final concentration of 2.5% DMSO

in the enzyme assay mixture. Al1 samples were assayed in duplicate.

The NAT 2 enzyme assay utilized an acetyl Coenzyme A regenerating system" to

maintain the AcCoA concentration at 100 PM, which is close to the physiological level of

the c~factor.~' The system was prepared with 5.4 mg of acetyl-DL-camitine and 1U of

carnitine acetyltransferase (E.C.2.3.1.7) per milliliter of NAT assay buffer. The NAT assay

buffer contained 250 mM triethanolamine-HC, 5mM EDTA and 5mM DTT, with pH

adjusted to 7.5. The DTT was added to complete the NAT assay buffer fi-om a 1M stock

solution when the regenerating system was prepared on the same day as the individual assay

was performed. Individual reactions with final volumes of 100 pl consisted of 10 pl of

Y--'---- --- -- , .. -- r- -- - - - r- . - - - - - - - - - - - - - - . - - -- - I

regenerating. system and 50 pl of the lysate. The lysate was used undiluted except for the

NAT2-SMZ assay in which the lysate was diluted five-fold with TEDK buffer. TEDK

consisted of 10 mM triethanolamine-HC1, 1mM EDTA, 50 mM potassium chloride and

1rnM DTT with a pH of 7.0. The DTT was added fkom a 1M stock solution shortly before

use. Individual samples containhg al1 components except the lysate were pre-incubated at

37 OC, and then the aliquot of lysate served to initiate the reaction. TEDK was substituted

for the lysate in blank reactions that were performed. Tubes were vortexed on an Fisher

Vortex Genie 2 and incubated in an Endocal RefXgerated Circulating Bath (Neslab) at 37

OC. Al1 hydroxylamine substrates were incubated for 10 minutes, while incubation times

for amine substrates ranged fkom 10 to 60 minutes. Reactions were terminated with the

addition of f O pl of 15% perchloric acid to precipitate the protein fkom the mixture. The

reaction tubes were then immediately fiozen in dry ice following termination of the reaction,

and stored at -80 O C until HPLC analysis. Prier to HPLC analysis of NAT2-sulfonamide-

hydroxylarnine reactions, individual reaction tubes were thawed, vortexed and centrifbged

at 15000 rpm on an IEC Centra 4B centrifuge (International Equipment Company)

microcentrifuge for 4 minutes to pellet the precipitated protein. For the amine substrates, the

reactions were not individually vortexed, centrifuged and analyzed. Rather, al1 samples

fiom an assay were thawed, vortexed and centrifuged simultaneously, and then WLC

analysis was conducted using the previously described system in Dr. Grant's lab which

utilized an auto sampler.

HPLC Quantitation

High performance liquid chrornatography was used to quanti& the product peaks

for both the NAT2-sulfonamide-hydroxylamine and sulfonamide reactions. Substrates and

product standards were used to determine the appropriate HPLC mobile phase conditions.

For the sulfonamide-hydroxylamine reactions, sensitivity factors with units of Absorbance

Unitslnrnole (AU/nmol) were determined for both the substraies and products by repeated

injection of known quantities of the compound ont0 the HfLC under the same conditions

which were to be used to assess the enzyme reactions. These sensitivity factors were used

to determine the amount of product produced in a reaction. For the sulfonamide substrates,

a standard containing known quantities of substrate and product was used in order for the

computer to calibrate the areas of these peaks for each individual assay. This calibration

allowed the direct conversion of peak areas to nmoles on the chromatogram. Standard

samples were dissolved in 2.5% DMSO.

For the NAT2-sulfonamide-hydroxylamine reactions, 50 pl of the supernatant fiom

each stopped reaction was individually injected ont0 a reverse phase Ct8 UBondpack

column (8 mm x 10 cm). Absorbance was measured at 254 nm at an Absorbance Units

Full Scale (AUFS) value of 0.300. Utilizing a flow rate of 2mVmin, various combinations

of acetonitrile and perchlorate buffer (20 mM sodium perchlorate, pH 2.5) mobile phases

were used to resolve the substrate and product peaks. Isocratic systems used between 13

and 35 percent acetonitrile with 65 to 87 percent perchlorate buffer, depending on the

substrate. The resulting retention times were approximately 4 minutes for the

hydroxylamine and 8 minutes for the acetylated product.

The HPLC of the NAT2-sulfonamide reactions was conducted using the previously

described HPLC system. Utilizing an autosampler, 50 pl of the supernatant fiom each

stopped reaction was injected ont0 a Beckman Ci8 Ultrasphere column. Absorbance was

measured at 250 n . at an Absorbance Units Full Scale (AUFS) value of 0.300. The system

used a flow rate of 2mllmin. The mobile phase consisted of 10% acetonitrile and 90%

perchlorate buffer for al1 sulfonamide substrates except sulfanilanilide, for which a mobile

phase containing 30% acetonitrile and 70% perchlorate buffer was used. Retention times

were approximately 4 minutes for the substrates and 6 minutes for the products, except for

sulfanilanilide, for which the correspondhg retention times were 2.4 and 2.2 minutes.

-

sulfonamide-hydroxylamine reactions, the areas were converted to nmoles, and then the

rates of the enzyme reactions were calculated with units of nmol/min/rng protein. For the

NAT2-sulfonamide reactions, the data was generated in units of nmoles. Non-linear

regression was used to fit the data to a c w e of reaction rate vs. substrate concentration.

The maximal enzyme velocities (V,) and the apparent Michaelis constants (Km) were

estimated using the Michaelis-Menton equation (v = V-*[S]/K,+[S]) and the curve-fitting

algorithm of ~rafit@ .

The NAT2 enzyme assays were performed using the previously described NAT

enzyme assay system. A range of substrate concentrations were incubated with wildtype

recombinant E. coli NAT2 enzyme Iysate, and the amount of product produced was assessed

by HPLC . Reactions at each substrate concentration were performed in duplicate.

Independent assays were performed at least twice. Product standards were used to

detennine sensitivity factors with units of AU/nmol for the acetyiated sulfonamide-

hydroxylamines and sulfonarnides. The protein content of the NAT2 E coli lysates was

assessed using the method of ~radford." The amount of product produced was converted to

a rate with the units of nmoi/&mg protein. The substrate concentrations and rates were

then used to construct reaction rate vs. substrate concentration c w e s fiom which the values

of Km and V,, were determined. The assay system was first tested with S M 2 as a substrate

to ensure the resulting Km and V,, values were comparable to those previously determined

for NAT2-catalyzed acetylation of SMZ using the same wildtype recombinant enzyme and

essentially the same method as described.

I) Studv of NAT2-catalvzed Acetylation of Suifonamide-hydroxvlamine Substrates

Although NAT2 is capable of tramferring the acetyl group fiom AcCoA to both the

nitrogen and oxygen atom of aromatic hydroxylamines, only the latter was observed for the

sulfonamide-hydroxylamine (SULF-NHOH) substrates studied. The first substrate which

was investigated was SMZ-NHOH, which was used to detennine the reaction conditions for

the rest of the SULF-NHOH substrates.

Determination of an Appropriate Incubation Time for SMZ-NHOH

In order to establish an appropriate incubation time for the SMZ-NHOH, 200 pM

SMZ-NHOH was incubated with NAT2 for times ranging fiom 2 to 60 minutes. It was

rather that the rates decreased with time (Figure 11). Because the NAT2 enzyme is known

to be stable for times much longer than one hour.4' instability was not deemed to be

responsible for this rate decrease. Moreover, there was less than 15% conversion of the

substrate to product did not appear to occur, so that depletion of the substrate availabie for

catalysis should not have significantly altered the rate of the reaction. Along with the

product SMZ-N-acetoxy (SMZ-NHOAc) which produced a peak on the HPLC

chromatogram at 6.6 minutes, another peak at 3.8 minutes continued to increase with the

incubation tirne. This retention time corresponded to that observed for the amine SMZ. At

sixty minutes, the areas of these two peaks were almost equivalent. In order to investigate

the cause of this, the stability of SMZ-WOAc in the assay solution was examuied.

Figure 11: Rate of NAT2-catalyzed conversion of SMZNHOH to SMZ-NHOAc at incubation times 2 to 60 minutes

41

incubation time (min)

As previously described, the NAT enzyme assay utilizes buffers in the lysate and

regenerating system in order to maintain the pH at approximately 7. The stability of SMZ-

NHOAc was examined in pH 7 phosphate buffer, and in NAT assay buffer with and without

the 5 m M DTT. SMZ-NHOAc was incubated in the appropriate buffer at 370C and HPLC

of the solution was conducted after 0, 30 and 60 minutes. From the data obtained fiom the

HPLC chromatograms, the major peaks corresponded to SMZ-NHOAc, SMZ and SMZ-

NHOH. The results of this experiment are listed in Table 1.

Table 1: Incubation of SMZ-NHOAc in phosphate and NAT assay buffer with and without DTT

A Phosphate

Buffer

B NAT assay

Buffer without DTT

C NAT assay

Buffer 1 with 5mM DTT 1 "- Present as an impurity in SMZ-NHOAc

Incubation Time (min)

O 30 60

O 30 60

SMZ-NHOAc

(nmoles)

3.14 2.95 1.94

3.30 2.32 1.65

In phosphate buffer, SMZ-NHOAc appears to be relatively stable for 30 minutes.

At 60 minutes, approximately 60% of SMZ-NHOAc remains, while about 10% has been

converted to the hydroxylamine and also to the amine. About 20% is unaccounted for. In

the NAT assay buffer without DTT, 20% of the SMZ-NHOAc has been converted to the

hydroxylamine and 70% of SMZ-NHOAc remains at 30 minutes. At 60 minutes, only 50 %

of the SMZ-NHOAc remains, while the arnount of SMZ remains relatively unchanged.

Again, there is material that is not accounted for. When 5 mM DTT was added to the NAT

(nmoles) (nmoles)

- - NHOH. At 30 minutes, there is an increase in the amount of SMZ which corresponds to

about 16% of the SM.-NHOAc originally in solution, and SMZ-WOH contributes 2 1% to

the 36% drop in the amount of SMZ-NHOAc. At 60 minutes, only 33% of the SMZ-

NHOAc remains, and an almost equivalent amount of SMZ and SMZ-NHOH are produced.

This result indicates that when SMZ-NHOH was incubated in the enzyme assay for 60

minutes, the SMZ-NHOAc product was being converted back to the hydroxylamine as well

as to the amine. As shown by experiment B of Table I , SMZ-NHOAc is converted back to

the hydroxylamine by the NAT buffer. One possible explanation for this is that the amine in

the buffer catalyzes the hydrolysis of the ester. Adding DTT increases the amount of

conversion back to the hydroxylamine, and at the same time results in significant reduction

to the amine, Possible mechanisms for this effect are shown below in Figure 12. DTT is

necessary in order to maintain the cysteine sulfhydral groups in a reduced state. It was

therefore not possible to remove DTT îkom the reaction solution. hstead, the reaction times

were limited to ten minutes for NAT2-catalyzed acetylation of sulfonamide-hydroxylamine

substrates in an effort to minimize the conversion of product back to the substrate and to the

amine while still achieving adequate conversion for accurate HPLC detection.

HS -CH, I CH-OH

CH-OH I

HS-CH, Dithiothreitol (DTT)

ArNH, + S-s I I CH2 CH2 I I

CH-CH

I I OH OH

Protein Assays

Protein assays were performed as previously described using the method of

~ radford~ ' . The total amount of protein in the lysate was determined and used to express

the rates of enzyme catalysis in units of nmol/min/mg protein. Different batches of NAT2

enzyme lysate were used for different assays as listed in Table 2. Although the NAT2

lysates were prepared using the same method (Appendix B), the amount of protein was

found to vary slightly between the batches. The amount of NAT2 is assumed to be

proportional to the total protein concentration in the lysate.

assays

Protein Concentration

OwW

NAT2 Assays performed

0.90 SMZ-NHOH 1 SMZ-NHOH2

SPY-NHOH 1 SPY-NHOH2 SMZ-NHOH3

SD-NHOH 1 SD-NHOH2 SD-NHOH3 SD-NHOH4

SMR-NHOH 1 SMR-NHOH2 SMR-NHOH3 SMR-NHOH4

HPLC Conditions

In the reverse-phase HPLC method which was employed, more polar compounds

are eluted from the column first. In order of decreasing polarity and increasing retention

times, the order of elution for the substrates and product standards was usually sulfonamide-

hydroxylamine (SULF-NHOH), sulfonamide (SULF), sulfonamide-N-acetyl (SULF-NHAc),

and fmally sulfonamide-N-acetoxy (SULF-NHOAc). Under the HPLC conditions used for

the NAT2-sulfonamide-hydroxylamine assays, the components of the NAT enzyme assay

were eluted before any of the substrate or product peaks. Appropriate HPLC conditions

were detemined for the sulfonamide-hydroxylarnine substrates and suifonamide-N-acetoxy

- - - - factors which were considered when altering the acetonitri1e:perchlorate buffer ratios. The

mobile phases and retention times for the sulfonamide-hydroxylamines and the sulfonamide-

N-acetoxy compounds are listed in Table 3. A sample chromatogram for one reaction is

given in Figure 1 3.

Table 3 : HPLC conditions and retention times used for NA=-suifonamide-hydroxylamine assays

Substrate SMZ- SD- SPY- SPE- NHOH NHOH NHOH NHOH

Acetonitrile 19 17 15 19 Y0

Perchlorate 81 8 3 8 5 81 Buffer Y0

Retention 3.9 3.7 3.8 4.8 Time

SUL& NHOH

Retention 6.6 5.6 8.2 8.6 Time

SULF- NHOAc

(min)

SMR- SAA- NHOH 1 NHOH

This sample chromatogram is fkom an incubation of 400 pM SAA with NAT2 for ten minutes. The peaks at 4.60, 5.88, and 8.70 minutes correspond to SAA-NHOH, SAA, and SAA-NHOAc respectively .

The purity of the substrates and product standards were assessed by TLC (Appendix

A) and by HPLC. There were usually amine impurities in the hydroxylarnine s*strates

that accounted for less than 5% of the total compound. Approximate impurity contents of

the SULF-NHOHs are listed in Table 4.

Table 4: Approximate impurity contents of SULF-NHOH substrates

The amine SPE was not the only irnpurity present in the SPE-MIOH sample. There

SPE-IWOH SPY-NHOH SAA-MHOH

were peaks on the HPLC chromatogram which correspond to what may be the nitro and

nitroso fonns of SPE. The combined amount of impurities however, appear not to be more

than 5 %, and the SPE-NHOH was pure by TLC (Appendix A).

-95 98 98

The SULF-NHOAc compounds were considered pure by TLC (Appendix A), but

impurity peaks were visible by HPLC. Based on the retention times, the impurity peaks

2 2 2

were attributed mainly to SULF-NHOH and SULF. The approximate percentages of

3 O O

impurities for al1 of the product standards are listed in Table 5. Like the SULF-NHOH

substrates, the level of impurities was low. These low levels of impurity in the standards

did not significantly affect the sensitivity factors necessary for quantifjing the amount of

product produced in the enzyme reactions.

Kine tics

Al1 assays of NAT2-catalyzed O-acetylation of the SULF-NHOHs studied were

Product Standard

performed using the same reaction conditions. The reactions used the enzyme assay system

previously described with 10-1 5 final substrate concentrations ranging fiom 30 to 1000 PM.

Al1 samples were assayed in duplicate using undiluted E. coli NAT2 lysates. Incubation

times were limited to ten minutes for al1 assays. After terminating the reaction with 10 pl

of 15% perchloric acid, the reactions were stored at -80 OC until HPLC analysis. A volume

of 50 pl of the reaction mixture was injected onto the HPLC and the chromatograrns were

analyzed to measure the amount of product produced. The factors which varied in these

experiments were the lysate batches (see Table 2) and also the HPLC conditions which were

% -NHOAc

used to quantifi the product peaks (see Table 3).

From the areas of the peaks on the HPLC chromatograms which corresponded to the

O-acetylated products, the number of moles produced by NAT2 were calculated using

independently detennined sensitivity factors (AU/nrnol). The number of nmoles were then

converted to rates of nmol/min/mg protein using the incubation time of ten minutes and the

amount of protein in the reaction mixture. The data was then used to construct curves of

O reaction rate vs. substrate concentration using the curve-fitting algorithm of the Grafit

software program which fitted the curves to the Michaelis-Menton equation. The data for al1

assays is contained in Appendix C. A sample reaction rate vs. substrate concentration curve

is shown below in Figure 14.

% - NHOH % NH2

NAT2-SMZ-NHOH Assay #1

[SMZ-NHOHJ (PM)

Problems with NAT24uifaaerine-hvdroxvlamine Assavs

The NAT2-catalyzed acetylation of the sulfonarnide hydroxylamine SPE-NHOH

was perfomed but is not reported. Due to problems with the synthesis of the SPE-NHOAc

product standard, the amount of product produced could not be quantified.

The apparent kinetic parameters of the NAT2-SMZ-NHOH assays are listed in

Table 6. The average V, value fiom the three independent assays was 6.35 f 0.51

nmoI/min/mg. The Km values averaged to 173 + 2 1 FM. The average V,,,/Km value was

36.7 x 10".

Table 6 : Results of the NAT2-SMZNHOH Assays

Assay #

1

2 5.89 0.27 158 22 37.28

I 3 I 6.27 I 0.23 1 I 18 i 38.47 I

h a

(nmoY &mg) 6.90

Std. En-.

0.20

K m (PM)

197

Std. Err.

15

V,U& (X 10.~)

35.03

The apparent kinetic parameters of the NAT2-SMR-NHOH assays are Iisted in Table 7.

The average V, value fiom the three independent assays was 6.06 f 2.07 nmol/min/mg.

The Km values averaged to 940 f 339 pM. The V,,/K, average value was therefore 6.45 x

Table 7 : Results of the NAT2-SMR-NHOH assays

LdL (X IO-')

6.93

6.1 1

6.46

Km (W)

610

924

1287

Std. En.

0.30

0.94

0.92

Assay #

1

2

3

Std. Err.

75

250

211

L a

(nmov minlmg)

4.23

5.65

8.3 1

The apparent kinetic parameters of the NAT2-SDZ-NHOH assays are listed in

Table 8. The average V,, value fiom the four independent assays was 8.22 It 1.50

nmoVminhng. The K, values averaged to 1244 f 2 10 pM. The V-Km average value was

therefore 6.6 1 x 1 O-'.

Table 8 : Results of the NA=-SDZ-NHOH assays

Assay #

1

2

3

4

Vm ( n m o ~ &mg)

9.32

9.57

7.56

6.42

Std. Err.

0.50

2.17

2.26

0.99

Kr" (PM)

1299

1433

1299

943

Std. En.

1 03

467

577

236

%I~/K, (X IO-))

7.17

6.68

5.84

6.8 1

The apparent kinetic parameters of the NAT2-SPY-NHOH assays are listed in Table

9. The average V,, value fiom the four independent assays was 5.80 + 1.18 nrnovmidmg.

The K, values averaged to 128 f 45 ~.LM. The Vm,/Km average value was therefore 39.68 x

10-~.

Table 9 : Results of the NA=-SPY-NHOH assays

Assay #

1

V m ( n m o ~

midmg) 4.69

Std. ER.

O. 15

K m (PM)

112

Std. Err.

10

v,~/Kln (X 10-~)

4 1.88

The apparent kinetic parameters of the NAT2-SAA-NHOH assays are

The average V,, value fiom the two independent assays was 2.65 f

listed in Table 10.

0.2 1 nrnol/min/mg.

The Km values averaged to 73 f 5 W. The V,&, average value was therefore 36.30 x

1 O-3.

Table 10 : Results of the NAT'-SM-NHOH assays

Assay #

1

2

v,, (nmo~

midmg) 2.50

2.79

Std. Err.

0.08

Km (PM)

76

Std. Err.

9

0.09

V,W/L (X 10-~)

32.89

9 69 40.43

Protein Assay

A single batch of recombinant E. coli NAT2 enzyme lysates was used for al1 assays.

The protein content of the lysate was measured to be 1.33 mg/ml using the previously

described method.

HPLC Conditions

The HPLC of the NAT2-sulfonamide substrate reactions was performed using the

previously described HPLC system in Dr. Grant's lab. A mobile phase consisting of I O %

acetoniûile and 90 % perchlorate buffex was sufficient to achieve adequate peak separation

for assessing the peaks in al1 reactions except for those assays using suifanilanilide as a

substrate. A mobile phase consisting of 30 % acetonitrile and 70 % perchlorate buffer was

used for SAA. Unlike the system used for the sulfonarnide-hydroxylamine assays, a

component of the enzyme assay system was eluted close to or partially coinciding with the

substrate or product peaks. This peak corresponded to AcCoA.

Puriiv of Substrates and Product Standards

Unlike the sulfonamide hydroxylamine substrates, many of the sulfonamides were

commercially available. Sulfapyridine, sulfamerazine, sulfarnethazine and sulfadiazene

were al1 purchased. These compounds were assessed for impurities by HPLC and appeared

pure. The sodium salts of sulfadiazene and sulfamerazine were also commercially available

and pure by HPLC. Sulfanilanilide and sulfaperine were synthesized and assessed for purity

by TLC (Appendix A) and HPLC. They too appeared to contain essentially no impurities.

The purity of the N-acetylated sulfonarnides was also evaluated by both TLC

(Appendix A) and HPLC and there were no significant impurity peaks.

The solubility of the sulfonamide substrates dictated the concentrations used for the

NAT2 assays. The sodium salts of SDZ and SMR were commercially available and

significantly more soluble than the sulfonamides alone, and were therefore used as

substrates for the enzyme reactions. The solubilites of SMZ and SPY in 25 % DMSO w ~ r e

sufficient to yield enough information about the kinetic parameters of the enzyme reactions.

Studies of the NAT2catalyzed acetylation of SPE and SAA however were limited by the

solubility of these substrates in 25 % DMSO.

The solubility of these substrates in alternative solvents was not explored due to the

effect other solvents have on the activity of NAT2. A study by Svensson and Ware revealed

that of 5 solvents studied at various concentrations, DMSO caused the smallest amount of

inhibition of NAT2 a c t i v i t ~ ~ . ~ ~ DMSO at a concentration of 2.5% still resulted in a drop in

NAT2 activity of approximately 20%, however alternative soIvents resulted in an even

greater magnitude of inhibition. The concentration of DMSO was not increased due to the

effect it would have had on the activity of NAT2, resulting in kinetic parameters which were

incomparable.

Kinetics

The assays of the NAT2-catalyzed N-acetylation of the sulfonamides studied were

performed using varyhg reaction conditions. Pilot assays were performed to determine

appropriate incubation times and lysate dilutions to ensure that a significant amount of the

substrate was not converted to product in order to ensure that initial rates could be

measured. The reaction times, substrate concentration ranges and lysate dilutions for these

assays are listed in Table 1 1.

1 Substrat. 1 Incubation time (min)

Substrate concentration I Lysate range Dilution

S M Z SMR SPE SDZ

Problems with NAT2-SPE Assavs

The sulfonamide sulfaperine (SPE) was used as a substrate for two NAT2 assays.

However, the results are not reported due to solubility limitations of this substrate in 25%

DMSO. The highest final substrate concentration possible was only 100 pM, which was

insufficient to define a reaction rate vs. Substrate concentration curve.

SPY SAA

10 30 60 30 15 30

(kW 50 - 1500 100 - 6000 30 - 100 50 - 5000 50 - 2000 50 - 500

5-fold

1) Sulfamethazine

The apparent kinetic parameters of the NAT2-SMZ assays are listed in Table 12 . The

average V,, value fiom two independent assays was 12.74 f 0.09 nmoL/Wmg. The Km

values averaged to 132 f 11 pM. The V,,/K, average value was therefore 96.52 x IO?

Table 12 : Results of the NA=-SM2 assays

Assay #

1

2 12.80 0.24 12 9 1 -43

Std. Err.

6

v m , (nQloi/

midrng )

12.67

v,m/Km (X IO-^)

102.18

Std. Err.

0.17

Km (PM)

124

The apparent kinetic parameters of the NAT2-SMR assays are listed in Table

average V, value f?om two independent assays was 8.41 k 0.25 nmol/rnin/mg.

values averaged to 1 1 14 k 45 @M. The V,&, average value was therefore 7.55 x

Table 13 : Results of the NA=-SMR assays

13. The

The Km

1 O-3.

Assay #

1

2

Std. Err.

O. 12

0.2 1

vma (I~MOV

midmg)

8.23

8.58

Km (W)

1 082

1145

Std. Err.

46

79

V ~ K I I (X loJ)

7.6 1

7.49

The apparent kinetic parameters of the NAT2-SDZ assays are listed in Table 14. The

average V, value fiom two independent assays was 4.84 + 0.24 nmol/min/mg. The Km

values averaged to 32 12 f 170 piid. The V,,,JK,,, average value was therefore 1.5 1 x 1 O".

Table 14 : Results of the NA--SDZ assays

~ u K ~ (X 10-~)

1.50

1.51

Assay #

1

2

v- (nmoll

midmg )

5.0 1

4.67

KIT, (PM)

3332

3092

Std. Err.

0.22

0.25

Std. Err.

274

239

The apparent kinetic parameters of the NAT2-SPY assays are listed in Table 15. The

average V,, value fiom two independent assays was 8.89 f 0.65 nmol/min/mg. The Km

values averaged to 5 16 f 26 pM. 7'he Vm&Cm average value was therefore 1 7.23 x 10".

Table 15 : Results of the NA=-SPY assays

Assay # KM (nmoV

midmg)

Std. En. Kt, (kW

Std. Err. V d ' m (x 10.))

The apparent kinetic parameters of the NAT2-SAA assay is listed in Table 16 . The V,,

value was 2.52 f 0.29 nmoWmin/mg (fit error). The Km was 75 1 k 124 pM(fit error). The

Vm&, value was 3.357 x 1 05.

Table 16 : Results of the NA=-SM assay

Std. Err.

124

K m (w)

75 1

V m d K m

(X 10-~)

3.357

Std. En.

0.29

Assay #

1

v m a x

(nmo~ midmg)

2.52

In this study, we have investigated the effects of alterations in substrate molecular

structure on the NAT2-catalyzed scetylation of a series of sulfonamides and their Nd-

hydroxylamine derivatives. The effect of oxidation of the Nd-amine group to a

hydroxylamine, as well as methyl substitution in the NI-pyrimidine ring, and altering the NI-

substituent fiom a pyrimidine to a pyridine or a benzene ring on the apparent kinetic

parameters V,, and Km, as well as V,,/Km were demonstrated.

The sulfonamides considered may be divided into 2 series as follows. Series 1

includes substrates which differ in the number of rnethyl groups in the pyrimidine ring.

Series la consists of sulfonamide-hydroxylamine derivatives SMZ-MIOH, SMR-NHOH,

and SDZ-NHOH, and Series l b consists of sulfonamides SMZ, SMR and SDZ. Series 2

contains substrates that differ in the number of nitrogens in the NI-substituent. Series 2a

consists of SDZ-NHOH, SPY-NHOH and SAA-NHOH, and Series 2b consists of SDZ,

SPY and SAA.

The Nd-NH2 to NNOH conversion resulted in substrates for which NAT2 was

generally more specific. This observation is based on the apparent Km values for the

substrates in both Senes 1 and 2. The apparent Km values also regularly decreased for both

the sulfonamide-hydroxylamines and sulfonamides in both Series 1 and 2 as the number of

methyl groups increased and the number of nitrogens decreased respectively. The apparent

V,, values increased with the number of methyl groups and nitrogens in the NI-ring for

both Senes lb and 2a. The ratios of V,&, displayed a clear trend of increasing with the

number of methyl groups only for Series Ib. The apparent kinetic parameters for al1 the

NAT2 reactions with both senes of substrates are contained in Table 17.

Series

1 a

lb

2a

In general, Km values may be treated as overall dissociation constants of al1 enzyme-

bound species in a reaction." The apparent Km values may therefore be interpreted as the

binding affinities of NAT2 for the substrates studied. Ratios of the apparent Km value of

the sulfonamide-hydroxylamine to that of the parent sulfonamide for al1 substrates are

illustrated in Figure 15. With the exception of SMZ-NHOH:SMZ, NAT2 displayed a

greater affinity for each sulfonarnide-hydroxylamine substrate when compared to the

sulfonarnide. This may be a result of the possible addition of another hydrogen bond

1

Substrate

SM'-NHOH

SMR-NHOH

SDZ-NHOH

S M Z

SMR

SDZ

SDZ-NHOH

2 b

I

Vmax

6.35

6.06

8.22

12.74

8.4 1

4.84

8.22

SPY-NHOH

SAA-NHOH

SDZ

SPY

SAA I

Km

173

940

1244

132

11 14

3212

1244

Vmax/Km (X 10-~)

36.71

6.45

6.6 1

96.52

7.55

1.51

6.6 1

5.08

2.65

4.84

8.89

2.52 I

128

73

32 12

5 16

75 1 I

39.68

36.30

1.51

17.23

3.36 I

. -

II metabolic enzyme, it rnay follow that the Phase 1 oxidation of the parent sulfonamide not

only makes the sulfonamide more water-soluble, but also increases the affinity of NAT2 for

the metabolite, demonstrating a cornplimentary combined effort of Phase 1 and Phase II

metabolism.

Figure 15: Ratio of apparent Km values of SULF-NHOH to SULF substrates in Series 1 and Senes 2

SM2 SMR SDZ SDZ SPY S M

Series 1 Series 2

Another trend was observed when the effect of molecular structure on the apparent

Km values for the NAT2 reactions were examined. With the exception of SAA in Series 2b,

as the number of methyl groups on the pyrimidine ring increased, and the number of

nitrogens in the ring decreased, the apparent Km values decreased. Figures 16 and 17

illustrate these trends for Series 1 and Senes 2 substrates respectively. These trends may

indicate that NAT2 displays a higher affinity for sulfonarnides which have more electron-

rich ring systems.

decreasing #of methyl groups i

- decreasing #of methyl groups -

SMZ- SMR- SDZ- SMZ SMR SDZ NHOH NHOH NHOH

NAT2 Substrate

Figure 17 : Apparent Km values for Seriea 2 substrates

4000 . I I I 1 1 decreasing #of niaogens - - -

SDZ- SPY- SAA- SDZ SPY SAA NHOH NHOH NHOH

NAT2 Su bstrate

acetylation by NAT2 of the sulfonamides in Series l b increase with the number of methyI

groups in the pyrimidine ring." The apparent maximal rate of NAT2 catalyzed acetylation

of these same substrates, V,, was also found to follow the same trend, although this effect

was not seen with the corresponding suifonamide-hydroxylamines of Series la. In series 2a,

the apparent V,, values increased with the number of nitrogens in the Ni-ring; this effect,

however, was not seen is Series 2b. The apparent V,,,, values for both Series 1 and Series 2

substrates are illustrated in Figure 18 and Figure 19 respectively. Overall, V,, changes by

less than one order of magnitude for the 10 compounds studied in this work, and considering

al1 of the compounds, the trends would have to be regarded as being irregular. It can also be

noted that it is unlikely that the biological rate of acetylation will reach V,, due to the

lower physiological concentrations of these drugs.

Figure 18 : Apparent V,, vrluem for Serien 1 substrates

- SMZ- SMR- SDZ- SM2 SMR SDZ NHOH NHOH NHOH

SDZ- SPY- S M - SDZ SPY SAA NHOH NHOH NHOH

NAT2 Substrat8

The ratio of the apparent kinetic parameters V,K, rnay be used as a relative

measure of the specificity constant kJC,. Of the NAT2 reactions studies, a clear trend

appeared only in Series lb, narnely SMZ, SMR and SDZ. For these substrates the V,&,

ratios increased dong with the number of methyl groups. This also follows the pattern

observed by Vree and coworkers of the rate of absolute acetylation of these sulfonamides.

The ratio V,,/K, is indicative of the initial rate of catalysis at low substrate

concentrations (slope of initial rate). This ratio may also be used to determine the catalytic

specificity of an enzyme for cornpeting substrates at any substrate concentration. Graphs of

reaction rate vs. substrate concentration up to 200 pM are illustrated in Figures 20 and 21

for Senes 1 and Series 2 substrates respectively. At these concentrations, it is evident that

the catalytic specificity of NAT2 for the substrates is determined by the magnitude of

V a . Under physiological conditions of d m g dosage, it is unlikely that most of these

compounds will achieve concentrations necessary to reach or surpass the Km. Therefore,

occur in vivo.

Figure 20 : Rate vs. substrate concentration curve for Series 1 substrates

O 50 100 150 200

[Substrate] (FM)

Figure 21 : Rate vs. substrate concentration curve for Series 2 substrates

- - -

- - SMZ-NHOH

- SDZ-NHOH

- - SMR-NHOH

- SMZ

- - - - SMR

- - - - SDZ

-- SDZ-NHOH

- SPY-NHOH

- - SM-NHOH

l m - - - SDZ

- - - - S f Y

O 50 100 150 200

[Substrate] (FM)

AU- r r u u u l u r i v r r iiuii iiiu v i u rvub a u uiv b u v r - w i i rvi u u v v i w u v w - -6 concentration (in plasma) to half of the original value."his parameter is dependent on

many different factors including absorption, excretion and protein binding of the dnig.

Values in man were reported for SMZ, SMR, SDZ and SPY by Vree and ~ekster."

Comparing the elimination half life (Tln) of these sulfonamides in fast acetylators to the

apparent specificity constants revealed a relative correlation between the time necessary for

this elimination and the constants. The elimination half-life of the h g is seen to decrease

as the apparent specificity constant of the enzyme increases. Since these specificity

constants may be considered measures of the initial rates of acetylation by NAT2, this

correlation suggests that the rates observed in the present study are indicative of the half life

of elimination of these drugs in vivo.

Table 18 : Comparison of the elimination half-lifes to the apparent specificity constants for SMZ, SPY, SDZ and SMR

Although the apparent kinetic parameters attained do show some defuiite trends and

c m be considered meaningful rneasurements of the NAT2-catalyzed acetylation of the

substrates studied, the sources of error in the methods used to assess these reactions rnust be

addressed. These errors mise £rom the substrate concentrations used to define the reaction

Sulfonamide

S M Z

SPY

SDZ

S M R

(hours)

2

5

11

12

Vmax/Km (X 10s)

96

40

7.55

1.5

NA ï 2 E. coli lysate batches used in the assays.

Assays were performed using various incubation tirnes and substrate concentration

ranges. The sulfonamide-hydroxyiamine substrate assays used reaction times of ten minutes

and final substrate concentrations ranghg fiom 30 to 1000 pM. Al1 substrates were solubIe

to 10000 pM in 25% DMSO. Ideally, the substrate concentrations should have significantly

surpassed the K, for each substrate. This would ensure that the substrate approached a

saturathg concentration, the reaction curve was fuily defined, and accurate apparent kinetic

parameters were attained. Even if the substrate concentrations were high enough to defme

the c w e around the Km where the data begin to dispIay saturation kinetics, the curve-fitting

software used should fit the curve to the Michaelis-Menton equation and determine apparent

kinetic parameters appropriate for the enzyme reaction. For the SM.-NHOH and SDZ-

NNOH substrates, the average Km values were 940 and 1240 pM respectively, while the

highest substrate concentration was 1000 W. The highest fuial concentration of SAA was

only 500 pM while the Km was determined to be 751 W. This assay was limited by the

solubility of SAA in 25% DMSO.

There does not appear to be much scatter in the data for separate assays of NAT2-

catalyzed acetylation of sulfonarnide substrates. The NAT2-sulfonamide-hydroxylamine

assays, on the other hand, often display scatter fiom the curve for many individual reaction

concentrations. Possible reasons for these variations in rate observed in the individual data

points for the hydroxylarnine assays include impurities in the substrates and the stability of

the substrates and products in the reaction solution.

As previousiy described, there were impuilties in the sulfonamide-hydroxylarnine

substrates which appeared to account for less than 5 percent of the total amount of

impurities were visible only by HPLC. Because these impurities seemed to be the

corresponding amine sulfonamide compounds, they too would be substrates for NAT2. This

would affect the observed rates of acetylation for the sulfonamide-hydroxylamines,

especially if the apparent specificity constant of the amine was considerably smaller than

that of the hydroxylamine.

The apparent specificity constants, Vmm/Km, for SMR-NHOH and S M . were

approximately the same. For the remaining sets of sulfonamides and corresponding

sulfonamide-hydroxylamines, there were more considerable variations in the values of

Vm,/K,. In general, at substrate concentrations over 400 pM, there were usually small,

detectable peaks with retention times which conesponded to the SULF-NHAc compounds.

Presumably, these SULF-NHAc compounds resulted fiom the NAT2-catalyzed acetylation

of the amine impurities. The areas of these peaks, however, did not represent a significant

conversion which may have interfered with the acetylation of the SULF-NHOH substrate.

At lower substrate concentrations as well, there was inevitable acetylation of the

sulfonamides by NAT2 coincident with acetylation of the sulfonamide-hydroxylamines.

The stability of the su1 fonamide-hydroxylamine substrates in 2.5% DMSO, the

concentration used for the assays, could be assessed by monitoring the peaks present when

sensitivity factors were determined over the course of one to two hours. The stability of the

sulfonamide-hydroxy1amines under the conditions of the NAT2 enzyme assays could be

assessed by analysis of the chromatograms of the blank reactions which were perforrned by

substitution of TEDK buffer for lysate. The sulfonamide-hydroxylamines appeared to be

stable in 2.5% DMSO for two hours, as neither an increase in the amount of sulfonamide

Mpurity nor the creation of new peaks were detected. The sulfonamide-hydroxylamines

minutes the substrates did not appear to undergo conversion to other compounds. This

assessment is based on the totaI nurnber of nrnoles present as product and substrate.

The sulfonamide-N-acetoxy products of the NAT2-sulfonarnide-hydroxylamine

reactions were found to be unstable in the reaction solution. As previously discussed for

SMZ-NHOAc, these products undergo conversion back to the sulfonamide and the

sdfonamide-hydroxylamhe under the conditions of the assay. This conversion has also

been found to occur ui vlho for sdfarnethoxamle-~-acetoxy,~' For al1 reactions of

sulfonarnide-hydroxylamines with NAT2, the reactions were fiozen at -80 OC following

termination with 10 pl of 15 % percldoric acid in an effort to prevent any loss of product

prior to HPLC quantitation. Even when stored at -80 O C however, there was approximately

a 4% Ioss in product per &y of storage. During the process of thawing and centrifugation,

and even elution fiom the HPLC column, there was also an inevitable loss of SULF-

NHOAc product due to this conversion. An effort was made to ensure that the thawing and

centrifugation times were equivalent to maintain consistency in the amount of time the

individual reaction tubes were allowed to remain at room temperature. Although the

instability of al1 of the SULF-NHOAc compounds were not examined to the same extent as

the SMZ-NHOAc cornpound, it is likely that al1 such compounds undergo the sarne

conversion, resulting in loss of quantifiable product. This may have caused variations in

the observed acetylation rates of reactions at individual substrate concentrations if the

reaction tube was allowed to remain at room temperature for a longer than average period of

tirne.

The NAT2 enzyme concentrations in the E. coli lysates were assumed to be

proportional to the protein concentrations of the individual lysate batches. Different batches

the amount of protein was quantified, this is not an absolute measure of the arnount of active

NAT2 protein in the lysate. Minor variations in the preparation of the lysate may have

altered the enzyme concentration. Although these variations in enzyme concentration would

not alter the apparent Km values, the limiting apparent V,,, values would be affected, and

thus so would V,JK,. Therefore, the V,, values must be interpreted cautiously.

The enzyme assay which was utilized in these experiments affected the apparent

V, values obsemed not only by the use of different lysate batches, but also by the use of

DMSO as a solvent necessary to solubilize the substrates. As previously mentioned,

although DMSO has been detennined to be the least detrimental solvent to the enzyme

activity of NAT2, it is still responsible for a 20 % drop in enzyme activity at a concentration

of 2.5%. The sulfonamides and sulfonamide-hydroxylamuie substrates are not readily

soluble in aqueous solution, and DMSO was necessary in order to solubilize the substrates

for the enzyme reactions.

The enzyme assay system used attempts to imitate conditions which are found in

vivo in human liver cells. Comparisons of Km and V,, kinetic parameters of NATs from

human liver cytosol vs. recombinant human NAT enzymes has revealed that the Km values

are maintained by the two enzyme sources, while the V,, values are higher for the

recombinant enzyme system used?' This suggests that the concentration in the lysates used

in the artificial system are higher than those found in vivo. So, although the expression

level of recombinant NAT2 in E. coli is not indicative of expression in human liver, the

relative affinities for the substrates studied should correspond to the native hwnan enzymes.

The apparent V,, values attained are useful then only in comparison of the rates of

acetylation of the various substrates by the recombinant NAT2 enzyme.

of the compounds studied suggest that the molecular structures of these sulfonamides and

sulfonamide-hydroxylamines affect the substrate specificity of NAT2. N4-hydroxylamine

derivatives generally displayed smaller Km values when compared to the respective parent

sulfonamides. Also, both increasing the number of methyl groups in the pyiimidine ring,

and decreasing the number of nitrogens in the NI-substituent resulted in increases in the

substrate specificity of NAT2. The V, values for al1 substrates ranged fiom 2.52 to 12.74

nmol/min/mg. in cornparison with the in vivo results previously demonstrated, both the

V, values and the V,& ratios were found to increase with the addition of methyl

groups to the pyrimidine Rng for the SMZ, SMR, SDZ series.

The results clearly demonstrate that NAT2 catalyzes the O-acetylation of the N4-

hydroxylamine derivatives of these sulfonamides. These hydroxylamine derivatives have

been postulated to play a role in mediating sulfonamide hypersensitivity reaction~.~'~~*"

Subsequent acetylation to produce the N-acetoxy metabolites may result in a more toxic

compound. The precise roles of these metabolites in mediating the adverse dnig reactions

associated with sulfonamide therapy have yet to be elucidated. Sulfamethoxazole and

sulfadiazene appear to be the most widely studied sulfonamides in this area presumably due

to their more widespread current clinical use compared with the other suifonamides in the

panel of compounds evaluated in the present study. As slow acetylators appear to be more

susceptible to the toxic effects of sulfonamide metabolism, the results of this study of the

poIymorphic substrates SMZ, S m , SDZ, and SPY may be usefd in future studies relating

to this issue.

The results of these experiments, in conjunction with similar studies of NAT2-

catalyzed acetylation of panels of closely related substrates, may help to determine the

structural features of NAT2 responsible for the observed trends in both apparent substrate

type catalytic specificiîy will be used in Dr. Grant's lab in order to compare the abiIity of

these enzymes to acetylate the substrates used in this study.

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Figurel: Structures of the compounds which were synthesized

SAA NO2 x = y= CH, R = NO2, R1=R2=R3 = H SAA NHOH x=: y= CH, R =NHOH, Ri=Rz=R3 = H SAA NHOAc x= y= CH, R =NHOCOC&, RI=&=& =H SAA NHz x=y=CH, R=NH2, R1=W&=H

SPY NO2 x=CH,y=N, R=NQ,Rl=W&=H SPY WOH x=CH, y = NI R= NHOH, R1=-& =U SPY NHOAC x =CH, y = NI R = NHOCOCH3, RI=&=& = H SPY N(Ac)OAC x =CH, y = N, R= N(COC&)OCOC&, RI=&=& = H SPY NHAc x=CH, y = NI R=NHCOCh, R1=R2=k = H

SDZ NO2 X=Y = N, R = NO2, R1=RpR3= H SDZ NHOH x = y = NI R = NiOti, RI =R2=R3 = H SDZ NHOAc x =y = NI R = N-(OCOCH3, Rl=R=& = H SDZ NHAc x =y = N, R = NiCOCH3, Rl=R2=& = H

SMR Nû2 x=y=N, R=NQ, Rl=CH3, &=&=H S M R N i W x=y=NR=NHOH,Rl=CH3,&=R=H SMR NHOAc x=y= N, R=NHOCûC%, Ri= Cl%, &=R3=tf SMR N(Ac)OAcx=y= N, R=N(COC%pCOC&, RI= CH3, R2433 =H SMR NHAc x=y = NI R = NHCOCk, Ri=CH3, R A 3 3 = H

SPE NO2 x=y=N,R=N02, &=C)-f3,Rl=&=H SPE NHOH x=y= NI R=NHOH, RF CH3, RI=& =H SPE NHOAC x=y = NI R= NHOCOCH3, &=CH;r, Rl=R3= H SPE NH2 x=y=N,R=NH2, R2=CH3, R1=&=H SPE NHAc x=y= NI R= NHCOCH3, R2=Ct(3, RI=5 =H

SMZN02 x=y=N,R=NO;!,I%=H, Ri=R3=Cb SMZNHOH x = y = N, R= NHOH, &=H, Ri=% =CH3 SMZ NHOAc x =y= NI R = NHOCOCH3, &=Hl Ri=&=Ci-b SMZ N(0H)Ac x =y= NI R = N(OH)COCk, R2=H, R i = k =CH3 SM2 NHAc x=y= NI R = NHCOCb, &=Hl Ri=R = C h

Synthesis of Nitrobenzenesulfonamides:

A mixture of 4-nitrobenzenesulfonylchloride (0.1 mol) and an equimolar amount of the

required amine (see Note 1) in dry pyridine (5.0 ml) was heated for 45 min at 60-70" and

then another 45 min at 105-1 10" (for maximum evaporation of pyridine). The resultant

brownish red crystalline mass* was treated with boiling acetic acid (50 d) and filtered

off. The residue was washed with acetic acid, HCI 1N (to remove unreacted h e ) and

frnally with water. The yellow mass was recrystallized fiom acetic acid to afford crystals

of nitrobenzenesulfonamides. The yield was in the range of 60 - 80%.

*To improve the yield and simplify the reaction procedure, the reaction mixture can be

poured on ice-water and the resultant solid can be washed with acetic acid, HCl IN,

acetone and finally with water

Note 1 : for SMZ: 2-amino-4,6-dime~ylpyrimidine

for SMR 2-amino-4-meaylpyrimidine

for SPE: 2-amino-5-methylpyrimidine

for SDZ: 2-aminopyrimidine

for SPY: 2-aminopyridine

for SAA: aniline

A solution of nitrobenzenesulfonamide (0.1 mol) in dry tetrahydrofuran (175 ml)

containing palladium on carbon 5% (50% wet W/W) was treated with a slow dropwise

addition of anhydrous hydrazine (1 -0-1.25 mol). To initiate the reaction, the mixture was

warmed to 40 '~ . The reaction was monitored by TLC using n-hexane and ethyl acetate

(4:l). The hydroxylarnine can be identified using iodine as a visualking agent which

reveals a speciai yellowish spot with brom boundaries. When the reaction was alrnost

complete, the mixture was filtered off and the solvent was removed under reduced

pressure. The crude product was dissolved in dichiorornethane or tetrahydrofuran,

washed with water, dried (MgS04), concentrated (10d) and then diluted with

cyclohexane to precipitate the hydroxylaminobenzenesulfonamides. The yield was in the

range of 65 - 90%. In a few cases, flash column chromatography was used to pur@ the

hydroxylamine. The elution solvent was varied initially fiom n-hexane:ethyl acetate

20: 1. The final eluant ratio was 5: 1.

Synthesis of N-Acetoxy Derivatives of Hydroxylaminobenzenesulfonamides (SULF-

NHOAc):

Acetylcyanide* (1.0 mmol) was added dropwise to a suspension of a solution of

hydroxylarninobenzenesulfonamide (1.0 rnmoi) and triethylamine (1.0 mmol) in

anhydrous tetrahydrofuran (50 ml) at O°C with continuous stirring for 45 min.

Additional triethylamine (10 ml) and acetylcyanide (20 ml) were added, and the

reaction was continued for 30 min. The liquid was concentrated under reduced pressure

and the acetoxy derivative was precipitated by diluting it with cyclohexane. Further

purification was done by preparative TLC; n-hexane and ethyl acetate (4: 1)

hydroxy laminobenzenesul fonamides

Synthesis of N-Acetyl Derivatives of Hydroxylaminobenzenesulfonamides (SULF-

NAcOH):

Acetyl chloride (76 ml, 1.0 m o l ) was added to a stirred suspension of

hydroxylaminobenzenesuifonamide (1.0 mmol) and sodium carbonate (1 .O g) in diethyl

ether (50 ml) at room temperature with continuous stuilng for 45 min. Additional acetyl

chloride (20 ml) was added and the reaction was continued for M e r 30 min. The

reaction was monitored with TLC, n-hexane and ethyl acetate (4: l), and a reddish-brown

spot was visudized using ferric chloride solution (10 %). The product was extracted

with NaOH IN, neutralized with dil. HCl and then back extracted with ethei. The

organic layer was dried (MgS04), concentrated and then diluted with cyclohexane to

precipitate the N-acetyl derivatives of hydroxylaminobenzenesulfonamides,

Synthesis of N-Acetyl Dervatives of Aminobenzenesulfonamides (SULF-NHAc):

To a suspension of aminobenzenesulfonamide (2.0 mmol) in anhydrous pyridine (5 ml),

acetic anhydride (2.2 m o l ) was added and stirred for 2 h at roorn temperature. The

reaction mixture was diluted with cold water (100 ml). The resdtant solid was filtered

off, washed with water, and recrystallized with absolute ethanol to obtain fine crystals of

the N-acetyl derivative of aminobenzenesulfonamides.

SULFANILANILIDE

SAA-NO2: 'H NMR (200 MHz DMSO-4) 10.62 (s, IH, disappeared in DzO), 8.37 (d, J

= 8.9 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 7.28 (m, 2H), 7.14 (m, 3H)

SAA-NHOH: 'H NMR (200 MHz, DMSO-4) 9.98 (s, lH, disappeared in D20), 8.97 (s,

lH, disappeared in DzO), 8.66 (s, 1H, disappeared in DzO), 7.54 (d, J = 8.6 Hz, 2H),

7.21 (m, 2H), 7.05 (m, 3l9,6.81 (d, J = 8.7 Hz, 2H)

SAA-NHOAc: 'H NMR (200 MHz, CDC13) 8.82 (s, lH, disappeared in D20), 7.69 (d, J

= 8.8 Hz, 2H), 7.24 (m, SH), 7.08 (m, 3H), 6.94 (d, J = 8.8 Hz, 2H), 2.28 (s, 3H)

S A A - m : 'H NMR (200 MHz., CDC13) 7.52 (d, J = 8.8 Hz, 2H), 7.25 (m, 2H), 7.05

(m, 3H), 6.56 (d, J = 8.6 Hz, 2H), 4.06 (s, 2H, disappeared in D20)

SULFAPYRIDINE

SPY- N02: 'H NMR (200 MHz, DMSO-4) 8.36 (d, J = 6.3 Hz, ZH), 8.10 (d, J = 7.8

Hz, 2H) 7.96 (d, J=6.2 Hz, 1H) 7.84 (t, IH), 7.30 (d, J= 8.5 Hz, IH), 6.88 (t, IH)

SPY-NHOH: 'H NMR (200 MHz, DMSO-4) 11.26 (s, lH, disappeared in D20), 8.95 (s,

lH, disappeared in D20), 8.67 (s, lH, disappeared in D-O), 8.07 (d, J = 4.4 Hz, 1 H),

7.70 (m, 3H), 7.12 (d, J = 8.5 Hz, 1H), 6.87 (m, 3H)

SPY-NHOAc: 'H NMR (200 MHz, DMSO-d6) 11.72 (s, lH, disappeared in D20), 10.40

(s, 114, disappeared in D20), 8.06 (d, J = 5.4 Hz, lH), 7.76 (m, 3H), 7.13 (d, J = 8.6 Hz,

lH), 6.98 (t, 3H), 2.23 (s, 3H)

SPY-N(Ac)OAc: 'H NMR (200 MHz, DMSO-d6) 8.10 (d, J = 5.5 Hz, lH), 7.9 1 (d, J =

8.8 Hz, 2H), 7.76 (t, IH), 7.66 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.4 Hz, lH), 6.87 (t, lH),

2.35 (s, 3H), 2.14 (s, 3H)

S P Y - W C : 'H NMR (200 MHz. DMSO-&) 1 1.80 (S. IH disappeared in D20), 10.18 (S.

lH, disappeared in DzO), 8.03 (d, J = 5.4 Hz, 1 H), 7.82 (d, J = 8.9 Hz, SH), 7.73 (m,

lH), 7.70 (d, J= 8.1 Hz, 2H), 7.13 (m, lH), 6.90 (d, J= 8.1 Hz, l m , 2.07 (s, 3H)

SULFADWINE

SDZ-NO2: 'H NMR (200 MHz, DMSO-d6) 12.25 (s, I H, disappeared in 40), 8.56 (d, J

= 5.8 Hz, 2H), 8.41 (d, J = 8.8 Hz, 2H), 8.24 (d, J = 8.8 Hz, 2H), 7.14 (m, 1H)

SDZ-MIOH: 'H NMR (200 MHz, DMSO-ci6) 11.50 (S. lH, disappeared in D20), 9.02 (s,

lH, disappeared in DzO), 8.69 (s, lH, disappeared in D20), 8.49 (d, J = 5.2 Hz, 2H),

7.77 (d, J= 8.8 Hz, 2H), 7.03 (m, lH), 6.85 (d, J = 8.8 Hz, 2H)

SDZ-NHOAc: 'H NMR (200 MHz, DMSO-4) 11 -52 (s, lH, disappeared in D20), 10.38

(s, lH, disappeared in D20), 8.51 (d, J = 5 Hz, H), 7.84 (d, J = 8.9 Hz, 2H), 7.05 (t,

lH), 6.98 (d, J= 8.9 Hz, 2H), 2.12 (s, 3H)

SD-NHAc: 'H NMR (200 MHz, DMSO-<Io) 11.70 (s, lH, disappeared in D20). 10.33 (S.

lH, disappeared in D20), 8.52 (d, J = 5.3 Hz, 2H), 7.92 (d, J= 8.9 Hz, 2H), 7.76 (d, J =

8.8 Hz, 2H), 7.08 (t, H), 2.08 (s, 3H)

SULFAMERAZINE

SMR-NO?: 'H NMR (200 MHz, DMSO-&) 12.30 (S. IH, disappeared in &O), 8.37 (m,

3H), 8.22 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 5.3 Hz, IH), 2.34 (s, 3H)

SMR-NHOH: 'H NMR (200 MHz,(DMSO-4) 11.25 (s, lH, disappeared in D20), 9.00

(s, 1 H, disappeared in D20), 8.69 (s, 1 H, disappeared in DzO), 8.33 (d, J = 5.1 Hz, t H),

7.76(d, J= 8.8 Hz, 2H), 6.85 (m,3H), 2.32 (s, 3H)

(s, 1 H, disappeared in DzO), 8.3 1 (d, J = 5.1 Hz, 1 H), 7.88 (d, J = 8.1 Hz, 2H), 6.97 (m,

3H), 2.32 (s, 3H), 2.24 (s, 3H)

SMR-N(Ac1OAc: 'H NMR (200 MHz, DMSO-4) 11.82 (S. lH, disappeared in D20),

8.38(d, J = 5.5 Hz. lH), 8.02(d, J = 8.8 Hz,2H),7.72 (d, J = 8.8Hz,2H),6.82 (d, J

= 5.3 Hz, lH), 2.37 (s, 3H), 2.33 (s, 3IQ2.15 (s, 3H)

SMR-NHAc: 'H NMR (200 MHz, DMSO-4) 1 1.62 (s, lH, disappeared in D20), 10.32

(s, 1 H, disappeared in D20), 8.32 (d, J = 5.1 Hz, 1 H), 7.92 (d, J = 8.1 &, 2H), 7.74 (d,

J = 8.3 Hz, 2H), 6.92 (d, J = 5.3 Hz, 2H), 2.32 (s, 3H), 2.08 (s, 3H)

SULFAPERINE

SPE-NO2: 'H NMR (200 MHz, DMSOC) 12.20 (s, lH, disappeared in D20), 8.42 (d, J

= 8.7 Hz, 2H), 8.31 (s, 2H), 8.22 (d, J = 8.6 Hz, 2H), 2.13 (s, 3H)

SPE-NH2: 'H NMR (200 MHz, DMSO-4) 12.20 (s, lH, disappeared in D20), 8.38 s,

2H), 7.62 (d, J = 8.7 Hz, ZH), 6.58 (d, J = 8.6 Hz, 2H), 6.0 1 (s, 2H disappeared in DzO)

2.12 (s, 3H)

SPE-NHOH: 'H NMR (200 MHz, DMSO-4) 1 1.25 (s, lH, disappeared in Da), 9.00 (s,

lH, disappeared in D20), 8.68 (s, lH, disappeared in D20), 8.34 (s, 2H), 7.77 (d, J = 8.8

Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 2.12 (s, 3H)

SPE-NHOAc: 'H NMR (200 MHz, DMSO-4) 10.42 (s, lH, disappeared in D20), 8.38

(s, 2H), 7.86 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 2.23 (s, 3H), 2.14 (s, 3H)

2-Amino-5-methylpyriniidine: 'H NMR (200 MHz, DMSO-d6) 8.14 (s, 2H), 6.18 (s, 2H,

disappeared in D20), 2.08 (s, 3H)

1 H, disappeared in DzO), 8.38 (s, 2H), 7.9 1 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.9 Hz,

2H), 2.12 (s, 3H), 2.08 (s, 3H)

SULFAMETHAZME

SMZ-Nq: 'H NMR (200 MHz, CDCl3) 8.36 (s, 4H), 6.66 (s, lH), 2.36 (s, 6H)

SMZ-WOH: 'H NMR (200 MHz, DMSO4) 11.25 (s, lH, disappeared in &O), 8.96

(s, lH, disappeared in DzO), 8.66 (s, lH, disappeared in DzO), 7.79 (d, J = 8.7 Hz, 2H),

6.84 (d, J= 8.8 Hz, 2H), 6.80 (s, lH), 2.22 (s, 6H)

SMZ-N(0HIAc: 'H NMR (200 MHz, DMSO-&) 1 1.72 (s, 1 H, disappeared in DiO),

10.86 (s, 1 H, disappeared in D20), 7.98 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H),

6.78 (s, lH), 2.26 (s, 9H)

S M Z - W C : 'H NMR (200 MHz, DMSO-d6) 11.50 (s, lH, disappeared in DzO), 10.35

(s, 1 H, disappeared in D20), 7.88 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 6.78 (s,

lH), 2.24 (s, 6H), 2.20 (s, 3H)

A 50 pl aliquot of Çesh overnight bacterial culture of DMG200 (pNAT2/XA90),

which produced the wild-type NAT2 protein was used to restart 5 ml of LB medium

containhg ampicillin (100 pg/ml). The culture was incubated at 37 OC and 280 rpm until

the OD600 was between 0.3 and 0.4. Afier three hours of expression directed by the

inducing agent IPTG = 1 .O mM), the cultures were removed fiom the incubator and

cenûifuged at 2050 x g for ten minutes at 4 OC in a swinging bucket centrifuge. The

supernatant was poured off and the tube inverted to for one to two minutes. The pellet

was resuspended, with 1/3 the volume of the original culture, in ice cold TEDK buffer

(1OmM triethanolamine-HCl, 1mM EDTA, 1 mM DTT, and 50 mM KCI, pH 7.0) using a

pipette.

The resuspended bacterial cells were tysed by sonication in a ice-water bath using a

Fisher Sonic Dismembrator Mode1 30C) (Fisher Scientific Limited, Ottawa, ON) at 60 % full

power for 2 x 20 seconds with 10 second breaks between sonications. This was usually

suficient to clear the suspension and release the soluble NAT protein fiom the bactenal

cells. The sonicated solution was then transferred to a 1.5 ml microcentrifuge tube and spun

at 18 320 x g for 5 min at 4 OC to pellet the cellular debris. The supernatant (the NAT2

lysate) was removed and transferred to a new tube.

The lysate was flash fiozen with liquid nitrogen and stored at -80 OC. Wild-type

NAT2 protein can withstand the rigors of a number of fieeze thaw cycles without a

significant loss in activity.

1) Tables of Data Measured for the NAT2-Sulfonamide-Hydroxylamine Assays

Table 1 : NATZ-SMZ-NHOH Assays

a) NAT2-SMZ-NHOH Assay # 1

Std. Err.

0.197

Kinetics

Vmax = 6.909

[SMZ-NHOH] (CLMI 30

v (nmol/minlmg)

0.797

[SMZ-NHOH] (PM) 30 30 30 50 50 80 80 100

100 140 140

180

180

250

250

300 300

400 400

500 600

600 800

800 1000

1 O00 1000

v (nmoI/min/mg)

0.861 1 .O70 0.860 1.560 1.420 1.990

2.040 2.280 2.300

2.730 2.600 3.360

2.600

3.570

3.660

3.680 3.800

3.600 4.1 10

6.1 20 5.100

4.810 4.720 4.730 5.220

4.530 4.830

Kinetics

Vmax = 5.886 Km = 158.48

Std. Err.

0.269

22.214

r [SMZ-NHOH] 1 v (nmollminlrng) 1 Kinetics 1 Std. Err. 1 (CLMI 30 0.890 Vmax = 6.269 0.229

a) NAT2-Sm-NHOH Assay # 1

[SMR-NHOHJ v (nmol/min/mg) Kinetics Std. Err. (PM) 50 0.308 Vmax = 4.233 0.297

( [SMR-NHOH] 1 v (nmol/min/mg) 1 Kinetics Std. Err. 1 (MW 30 0.125 V max = 5.648 0.941

[SMR-NHOH] (PM) 30

30

50 80

v (nrnol/rnin/rng)

0.188 0.179

0.376 0.568

Kinetics

Vmax = 8.307

Km = 1287.16

Std. Err.

0.923 21 1.36

a) NAT2-SDZ-NHOH Assay # 1

[SDZ-NHOH] (w) 30 50 50 80 80 100 100 140 140 180 180

Std. Err. v (nmo1lminlmg)

0.246 0.443 0.439 0.552 0.549 0.652 0.651 0.952 0.874

-

1.073 1 -090

Kinetics

Vmax = 9.319 Km = 1298.57

0.502 103.1 1

[SDZ-NHOH] (pM) 1 v (nmoVminlmg) 1 Kinetics Std. Err.

30 30 50 50 80 80 1 O0 100

140 140

180 180 250 250 300 300 400 400

500

500

600 600

0.21 8 0.257 0.330

0.364

Vmax = 9.572 Km = 1432.62

2.1 74 467.24

0.71 9 I 0.679 0.856 0.629 1 .O26 1 .IO8

1.179 1.202 1.314 1 A13 1.836 1.392 2.129 2.079 t .QI3

2.689

3.1 84 2.825

1 r

[SDZ-NHOH] (@)

30 30 50 50 80 80 1 O0

Kinetics v (nmol/minlmg) Std. Err.

0.234 0.235 0.414 0.366 0.494 0.525 0.696

Vmax = 7.564 Km = 1299.0

2.256 576.61

a) NAT2-SPY-NHOH Assay # 1

1 [SPY-NHOHJ (pM) ( v (nmol/min/mg) 1 Kinetics Std. Err.

30 30 50

1.130 1 .O78 1.409

Vmax = 4.690 Km = 112.174

0.151 10.119

~ P Y - N H O H ] (pM) 1 v (nmollminlmg) 1 Kinetics 1 Std. Err. 1 30

30

50

1 .O1 5

0.869 1.227

Vmax = 3.7074

Km = 84.66 0.21 5 16.137

Std. Err. [SPY-NHOH] (pM)

30 30 50

v (nmol/min/mg) Kinetics

O. 129 10.69

1

0.850 0.843 1.332

Vmax = 6.489 Km = 191.024

1

Std. Err.

0.176 33.169

1

[SPY-NHOH] (pM)

30 30 50 50 80 80 1 O0 100 140 140 180 180 250 250 300 300 400 1

v (nmol/min/mg)

0.855 0.860 1.477 1.401 1 -980 3.1 42 2.479 2.453 2.926 3.005 3.203 3.187 3.637 3.456 3.71 5 3.869 3.740 1

Kinetics

Vmax = 5.444 Km = 121.991

1

a) NAT2-SAA-NHOH Assay # 1

[SAA-N HOH] (w)

30

v (nmol/rnin/mg)

0.782

Kinetics

Vmax = 2.497

Std. Err.

0.0766

[SM-NHOH] (fl)

30

30

50

50

v (nmollminlmg) Kinetics Std. Err.

0.805

0.780 1 .O00

1.020

Vmax = 2.791 Km = 68.558

0.0861

9.134

. -'YU J'

F i m e 1 : NAT2-SMZ-NHOH reaction rate vs. substrate concentration curves

a) NAT2-SMZ-NHOH Assay # 1

[SMZ-NHOH] (FM)

b) NAT2-SMZ-NHOH Assay #2 8

-

6 - n

E -

A

A A- \ A A c .- A

E 4-

E C V

> 2-

O l ~ l ~ l ~ l ~ l ~ l ~ I l

O 200 400 600 800 1 o o O 1 2 0 0 1 4 0 0

[SMZ-NHOH] (FM)

[SMZ-N HOH] (PM)

a) NAT2-SMR-NHOH Assay # 1

8

7 -

6 - h

E 5- - C .- E 4- > O

E 3- Y

> 2-

1 -

O I I I I I I 1 I I O IO00 2000 3000 4000 5000

[SMR-N HOH] (PM)

b) NAT2-SMR-NHOH Assay #2

2000 aXX)

[SMR-NHOH] (PM)

a) NAT2-SDZ-NHOH Assay #1

[SDZ-N HOH] (fl)

b) NAT2-SDZ-NHOH Assay #2

4000 6000

[SDZ-NHOH] (FM)

[SDZ-N HOH] (CJV1)

d) NAT2-SDZ-NHOH Assay #4

4000 6mo

[SDZ-NHOH] (PM)

a) NAT2-SPY-NNOH Assay # 1

[SPY-NHOH]

b) NAT2-SPY-NHOH Assay #2

[SPY-NHOH]

[SPY-NHOH]

d) NAT2-SPY-NHOH Assay #4

[SPY-NHOH]

a) NAT2-SAA-M-IOH Assay # 1

b) NAT2-SAA-NHOH Assay #2

Table 6: NAT2-SMZ Assays

a) NAT2-SMZ Assay # 1

1 [SMZ] (m) 1 v (nmollminlmg) ( Kinetics 1 Std. Err.

b) NAT2-SMZ Assay #2

50 50

3.521 3.574

[SMZ] (pM) 50

Vmax = 12.665 Km = 124.105

v (nmollminlmg) 2.906

0.170 6.31 9

Kinetics Vmax = 12.802

Std. Err. 0.241

b) NAT2-SMR Assay #2

a) NAT2-Sm Assay # 1

Kinetics Std. Err. Vmax = 8.579 0.2085 Km = 1 144.953 79.2054

[SMR] (pM) 1 O0 1 O0 400

400 800

800 1200 1200

1600 1600 2000

2000

4000 4000

6000

6000

Kinetics

Vmax = 8.228 Km = 1082.1 27

v (nmol/rnin/mg) 0.630 0.664 2.041

2.082 3.585 3.600

4.345 4.075 5.018 4.947

5.495

5.478 6.483

6.503 6.882

6.859

Std. Err. O. 122 45.759

a j NAT2-SDZ Assay # 1

b) NAT2-SDZ Assay #2

[SDZJ (pM) 50 50

v (nmol/min/mg) 0.097 O.r)93

Std. Err. 0.252

238.979

[SDq (pM) 50

50

Kinetics

Vmax = 5.007 Km = 333 1 505

Std. Err. 0.224

274.243

v (nmollminlmg) 0.122

0.1 07

Kinetics Vmax = 4.666

Km = 3091.805 1 O0

1 O0 O. 164 I 0.166

a) NAT2-SPY Assay # 1

b) NAT2-SPY Assay #2

1 [SPY] (pM) 1 v (nmol/minlmg) 1 Kinetics 1 Std. Err.

Std. Err. 0.491 7

82.848

[SPY] (pM) 50

50

1 1 1 50 1 0.640 1 Vmax =9.352 0.526 1

v (nmol/min/mg) 0.652 0.660

Kinetics Vmax = 8.437

Km = 496.615

[SAAI (pM) 50 50 1 O0

1 O0

150 150 200

200

250

400 400 500

v (nmol/min/rng) 0.163

O. 160 0.263 0.253

0.41 8 0.41 1

0.554

0.526

0.684 0.840 0.91 1

0.99q

Kinetics

Vmax = 2.522 Km = 751.248

Std. Err. 0.292

123.626

Figure 6: NAT2-SMZ Reaction rate vs. substrate concentration curves

a) NAT2-SMZ Assay # 1

b) NAT2-SMZ Assay #2

Assay # 1

b) NAT2-SMR Assay #2

a) NAT2-SDZ Assay # 1

b) NAT2-SDZ Assay #2

a) NAT2-SPY Assay # 1

b) NAT2-SPY Assay #2

a) NAT2-SAA Assay

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