123 Notes, part 3 Enzyme mechanism - Examples of elucidationfst123.fst.ucdavis.edu/~garysmith/123Data.dir/Notes.dir/123_pt3.pdf · Here are some examples of the kind of information

123 Notes, part 3Enzyme mechanism - Examples of elucidationMajor principles

Binding site- the substrate must fit in the active site, so the active site must provide propercharge distribution, hydrophobic distribution, proper orientation, etc.

General acid/base catalysis- if a proton needs to be added or removed, there is usually ageneral acid or general base in the active site to do it (or to hold a water molecule to do it).

nucleophilic groups - when nucleophilic catalysis is in force, a nucleophile is provided.

other destabilization/stabilization of intermediates - metal ions may be present for binding,for polarizing bonds, etc. Some metals may add or remove electrons.

Major toolsThe tools for elucidation of the mechanism of action of enzymes we have discussed (there

are others) areKineticsIdentification of intermediates

kineticallyusing inhibitors,by exchange/partial reactions

pH-dependenceChemical modification(X-ray/NMR)

Here are some examples of the kind of information that has been used and of inferred mechanisms

Xylose isomeraseReaction: Catalyzes what appears to be a hydride transfer, or movement of a carbonyl group fromC1 to C2. Interconverts Xylose and Xylulose:

CH2OHHCOHHOCH

C OCH2OH

CH2OHHCOHHOCHHCOHC

OH

In solution, it probably looks like this:

OCH2OH

OH

OH

HO

H

H

H

HO

OHOH

HOHO

H

H

HH

H

H

FST 123 Part 3 G.M. Smith

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The enzyme also accepts glucose, which it converts to fructose (which could be but isn't called"gluculose"). So it can be used to produce high-fructose corn syrup from starch hydrolyzates.

How to compare substrates? Look at VmaxKM

; it measures both binding and turnover. Dr.

Whitaker has referred to it as a "specificity factor". Some people use kcatKM

, which is equivalent, but

also represents an apparent second-order rate constant (units: M-1s-1). See table 13.2 in Wong)

Sources/Size: Available from many microbial sources See table 13.1, P. 360 in Wong). Most aretetramers of ~120-180 kDa, with identical subunits (How could you tell? (measure Mr under nativeand denaturing conditions, end-group analysis to determine if the protomers are the same,separation (if necessary/possible) and sequencing). The E coli enzyme is a dimer of 44 kDa. X-raystructure shows two domains: 1n a/b8 barrel and a 5-helix-containing external loop. The symmetryindicates that the structure is a dimer of dimers.

T and pH: Most are relatively heat-stable; from B. stearothermophilus, it is especially so, with Toptof ~80°. Stable between pH 4 and 11. pHopt 7.5-8.0.

Other features: Requires a divalent cation Mg2+, Co2+, (Mn2+, Fe2+, Ni2+, Ca2+, Zn2+ and Cu2+

either don't work or produce less activity than Mg2+ or Co2+.). How to tell? Remove divalentcations using EDTA, then add back small amounts of soluble salts of other metal ions, check foractivity. How to compare? The metal stabilizes the protein's structure as measured by T-stabilityor resistance to denaturants.

What else does the metal do? Aid in binding? Aid in catalysis?Look at Vmax (Kcat) and KM with xylose as substrate using Mg2+, Co2+ and Mn2+.

Kcat KM Kcat/KM

Mg2+ Co2+ Mn2+ Mg2+ Co2+ Mn2+ Mg2+ Co2+ Mn2+

8.4 3.9 3.1 1.2 1.2 2.7 7636 3258 1137Apparently, both binding and catalysis are affected.

Inhibitors: Sugar alcohols.Active site:

Sequence information. If many sequences for evolutionarily related enzymes are available,residues that are important in binding and catalysis will be conserved. Of course, many others may alsobe conserved. Any residues we propose to be important in binding and catalysis had better beconserved, but this is not proof that they are important.

Physical studies of the metal-binding sites (techniques such as epr spectroscopy (of Cu2+,Mn2+, Fe3+, Ni2+), optical spectroscopy (especially for Co++ and Cu ion), resonance Ramanspectroscopy and Mossbauer spectroscopy (for Fe ion), X-ray absorption spectroscopy and EXAFS(extended x-ray absorption fine structure) can be used to study the coordination geometry, nature ofligands, etc. They are outside the scope of this course. X-ray diffraction and NMR spectroscopy canbe used to determine the entire 3D structure of enzymes. These techniques are also outside the scopeof the course. Some results will be given.

There are two metal binding sites per monomer. Both must be occupied for the enzyme to beactive. In all xylose isomerases studied, Mg2+ ion has the same Kd for both sites. In some, but not all


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of the XI's studied, Co2+ has different Kd's for the two sites: the two sites are therefore not identical.Site I: the metal is octahedral (6 ligands), with 4 ligands from the enzyme (two Glu-COO- and

two Asp-COO-). The other two sites are occupied by oxygen atoms from the substrate (or probablywater in the absence of substrate). The substrate is bound in the open-chain formand a threonine also binds the substrate, according to one study:

HCOHC

OH

CHCHOH

OCH2

OH

H

OC R

O

R CO

O

R CO

O

R CO

O

M

HCOHC

OH

CHCHOH

OCH2

OH

H

CH3

ThrCHOH

OC R

O

R CO

O

R CO

O

R CO

O

M

The second metal ion appears to bind to the carbonyl oxygen in another study.Chemical modification reveals an active-site histidine (other than the ligand).

Suggested mechanism (You can never prove a mechanism, just disprove other alternatives):Two reasonable hypotheses exist:1) proton abstraction leading to an enediol intermediate (like the triosephosphate isomerasemechanism)

BH+

B B

C CH

HHO O

RC C

H R

OHOH

C CHHROHO

:A-H:A H:A

and

2) a concerted hydride transfer (which requires metal ions and is therefore not possible intriosephosphate isomerase)


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OOH

OH OH

MM O-

HHO

HO OH

OH OH

MM

HH

Papain: A protease from papaya.

Reaction, Substrate specificity. Not very specific for any particular residues. Recentcrystallographic studies indicate binding sites for seven sidechains, each with its own preferences.Overall substrate specificity is an aggregate of these. Their relative importance and preferences can beassessed by comparing Kcat/KM for different substrates. Also hydrolyzes esters.

Sources/Size. Papaya latex of unripe fruit (note tissue specificity and growth-stage specificity. Manyrelated (but "different" enzymes are found in plants). From a GRAS source, widely used in the foodand beverage industry. 212 AA's, Mr = 23,350, monomer

T and pH: Quite T-stable at neutral-to-alkaline pH. Unstable at pH < 4. pHopt 5.5-7.5. kcat/KM showsa bell-shaped curve, optimum at pH 6 and pKa's of 4.3 and 8.5 (see below).

Other features. Seven Cys, three S-S bonds (leaves one free SH). Two clear domains, one of helices andcoil, the other of sheet.

Inhibitors: Thiol-specific reagents inactivate. Several classes of polypeptide inhibitors exist.

Active site: NMR is very useful for measuring the pKa of a Histidine sidechain. When the imidazolenitrogens are protonated, the ring is “more aromatic”, which gives the CH proton resonances chemicalshifts that are more like those of benzene (i.e., they move downfield at lower pH). The change inchemical shifts of the nitrogens themselves is even greater (see panel on the right).

NMR spectroscopy of papain shows that His is the residue with a pKa of ~8.5. Abnormallyhigh. The one free CysSH is conserved among several related proteins. Unusual result highlights ageneral principle: ionizing groups can influence one another if their pKa's are close. The Cys-SH


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ionizes to an anion, which stabilizes the charged form of the His imidazole (so it remains protonated ata higher pH than normal). BUT, during reaction, the Cys-S is blocked, being converted to a thioester,so the pKa of the His goes down. S-modified papain (can you design an affinity reagent that wouldinactivate it?) still shows a pKa near 4. In this form, the pKa of the Imidazolium is unusually low!

A thio-acyl intermediate has been inferred by several methods. The most direct is theobservation of the intermediate using 13C-NMR at very low temperature. The intermediate is trapped,because there is insufficient energy to convert the intermediate to the next intermediate in the sequence.

Suggested mechanismInferred from chemical and spectroscopic evidence and from the X-ray structures of enzyme-

substrate or enzyme-inhibitor complexes. The SH is already deprotonated and attacks the carboxylcarbon to form a tetrahedral intermediate; the tetrahedral intermed. collapses to expel the first product,which is protonated by the imidazolium, which is suddenly acidic because its ion-pair partner is nolonger charged:

SC

O-

NHR'

R

NNHH

R CO

NH R'

S-NN

HH

initial attack by deprotonated S tetrahedral oxyanion intermediate

The Imidazole H-bonds to a nearby water and makes it more nucleophilic. The water attacks thethioester to form a second tetrahedral intermediate, leaving the imidazole moiety protonated.

HO

H

NNH

+ H2N-R'

NNHH

SC

O-R

OHSC

RO

activation of water for hydrolysis cleavage of intermediate

When the second tetrahedral intermediate collapses, the S- is expelled, regenerating the initial state ofthe enzyme.

How else could you stabilize the intermediates during catalysis? The oxyanion species has acharge that neither substrate nor product has. Charged or H-bonding groups in this region of the activesite would also lower the energies. This portion of the active site is called the "oxyanion hole".

Chymotrypsin

PurificationProperties


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MrpIAA compositionActivation (ignore for now)

KineticsFirst, choose a substrate and an assay; there are many and studies date back to the 30's.

Possibilities include:•peptides of various composition and lengths- measure pH, appearance of new a-amino groups, loss ofsubstrate or appearance of product by HPLC, TLC, etc.•model compounds containing amide bonds (i.e., an amino acid in amide bond - measure ammonia usingNessler's reagent, etc.; an amino acid ester - measure pH, etc.)•model compound with a chromogenic leaving group (P1)

This is a two-substrate enzyme, since hydrolysis is a reaction between two molecules. But, oneof the substrates is water, whose concentration is immense (55.5M) and constant during a single runand it can't be varied in different runs. So we can consider the reaction to be a one-substrate reaction.Kinetics are simple: vary [S]o, measure Vo and plot 1/Vo vs. 1/[S]o. Calculate Km and Vmax andestablish that the enzyme does not exhibit allostery (it does not)

Substrate specificityWhat is "substrate specificity"? If substrate A has a lower Km than substrate B, then at equal

concentrations, A will occupy the active site more often than B and will stand a greater chance of beinghydrolyzed. However, if B has a higher Vmax than A, it will be hydrolyzed more readily than A once itbinds. So again, the measure of specificity is Vmax/Km. Do a double reciprocal plot for each kind ofsubstrate and compare them.

Use a series of the substrates and compare Vmax/Km. The things you could compare (i.e., varyby selecting different substrates) are chain length (analyze products: is it an exopeptidase, anendopeptidase or does it care?), the nature of P1 (the amino donor in the peptide bond) and the natureof P2 (the carboxyl donor in the peptide bond), and the nature of the bond between P1 and P2 (is it anesterase as well as an amidase?). The questions you would like to answer are where in a protein doesthe enzyme hydrolyze bonds and why AND what other kinds of bonds will it hydrolyze.

Answers: Chymotrypsin is specific for amides and esters in which the carboxyl group idprovided by an aromatic amino acid. Substituted phenylalanyl and tyrosyl esters and amides arehydrolyzed more slowly. Amides and esters of other large hydrophobic residues are also substratesbut are hydrolyzed slowly. The enzyme is not particularly selective for the nature of the alcohol oramine moiety.

Rapid kinetics (pre-steady-state)Occasionally, it is useful to do rapid-mixing experiments to see what happens in the very early

stages of the reaction, perhaps before the rate-limiting step in the overall reaction. Some of thesemethods are referred to as stopped-flow experiments.

One of the things you'd like to look for in a reaction with two products is an "initial burst"corresponding to the release of P1. An initial burst would occur if the overall RLS is the release of thesecond product. In this situation, the enzyme could not turn over again until P2 is released to generate


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free enzyme, so the steady state Vmax would correspond to the release step. But the pre-steady staterelease of P1 could be detected if it were in high enough concentration. If release of P1 is immediateand release of P2 is slow, we are claiming that the enzyme becomes saturated with P2 upon release ofP1. The amount of P1 released before steady state isestablished should be equal to the amount of enzyme present.

An initial burst is usually taken as indicative of acovalent intermediate, but is not proof of the existence of acovalent intermediate (just a stable one). If the release P2 is ratelimiting and corresponds to a hydrolysis of a covalent bondlinking P2 to the enzyme, then addition of a nucleophilestronger than water should enhance the rate of the overallreaction by regenerating free enzyme faster. N-Acetylphenylalanine methyl ester gives an initial burst equal tothe enzyme concentration; the poor substrate p-nitrophenylacetate does the same. Amides, however, do notshow an initial burst. Hydroxylamine accelerates the overallreaction during steady state.

Inhibitorsreversible

Substrate analogs without a hydrolyzable bond are inhibitors (e.g., indole, β-indolpropionate). Formyl-L-tryptophan and formyl-L-phenylalanine are also inhibitors: the use of 18Owater shows that the enzyme catalyzes the exchange of 18O into the inhibitor ("pseudosubstrate"),suggesting more strongly that they are bound at the active site. Isotope exchange is a “partial reaction”catalyzed by the enzyme

irreversibleDiisopropylfluorophosphate irreversibly inhibits the enzyme as do some acylating

agents. From sequence analysis, Ser 195 is found to be phosphorylated by the reagent.Toluenesulfonyl-L-phenylanylchloroketone (TPCK) is an affinity reagent that inactivateschymotrypsin and is found to modify His 57. Photooxidation of histidine also inactivates the enzyme

pH-dependenceamides

Hydrolysis of N-acetyl-tryptophan amide as afunction of pH at low [S] is bell-shaped, with pKa's near 7and 9. At high [S], the low pH ionization is also observed.Km is found to increase at pH 8.5, accounting for the basicionization. However, the change in activity on the basic sideis complex. It appears that the enzyme may undergo aconformational change with a pKa of 8.5-9.

The pH dependence can be dissected a bit more bylooking at Vmax and KM separately (next page).


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With amide substrates, it appears that the decrease in activity at alkaline pH involves an increase inKM.

The pH dependence of the reaction with ester substrates is different(!) This anomaly occursbecause the rate-limiting step of the reaction is different for amides and esters. Esters are actuallybetter substrates than amides, and form an acyl intermediate very quickly, so that the hydrolysis of theacyl enzyme intermediate is rate limiting. For amide substrates, formation of the acyl enzymeintermediate is rate limiting, so pH-dependence of amide binding influences the overall kinetics

StructureSequence and sequence comparisons (Bioinformatics)

Comparison of several sequences shows that certain residues are conserved. His 57 andSer 195 are among them.

Secondary Structure (circular dichroism, FTIR, Prediction...)


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X-rayactive siterole of sidechainsdeduced mechanism of action

A critical observation came from the X-ray structure by Blow and coworkers on crystals ofchymotrypsin and on crystals of chymotrypsin with a bound inhibitor, formyl-L-phenylalanine. Sincethis inhibitor is a pseudosubstrate, it was essentially certain that it binds at the active site. Locating itsbinding site amounted to locating the active site.

The key residues in the active site included the conserved Ser 195 and His 57. In addition, an asparticacid (also conserved) was found to be hydrogen bonded to the imidazole NH of His 57, and the otherimidazole nitrogen atom appeared to be H-bonded to the Ser 195 OH proton. These three residuesbecame known ad the catalytic triad, and residues at analogous positions are found in all serineproteases (e.g., trypsin, subtilisin, pepsin, elastase, α-lytic protease). There also followed acontrovwersy about whether the protons moved back and forth during the catalytic mechanism, whichis shown below.


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His 57

Asp 102

Ser 195

CO

HN

O

CNH

CH2

H

CHR

NH

HN NHO

OC

NH

Gly 193

Gly 193

NHN N

HO

OC

CNH

CH2

H

C

HN

OO

Ser 195

Asp 102

His 57

CHR

NH2(P1 released) Covalent intermediate

CO

O HNN

H O

NH

His 57

Asp 102

Ser 195

CHR

NHC

NH

CH2

H

CO

Gly 193

NH

Step 1

Step 2

Muscle Aldolase – Bernard Horecker’s mechanismAldolase catalyzes the cleavage of fructose-1,6-bisphosphate to dihydroxyacetone phosphate plusglyceraldehyde-3-phosphate in glycolysis, and the reverse of this reaction in glyconeogenesis. BernardHorecker found that the strong reducing agent sodium borohydride irreversibly inactivated the enzymein the presence of either DHAP or F-1,6-bisP. If the borohydride was tritiated, tritium appeared in theinactive enzyme. The tritiated enzyme could be cut enzymatically and the resulting peptides could bemapped to determine where the tritium was attached. The tritium and a reduced derivative of the


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DHAP were attached to the ε-amino group of a lysine, implying the presence of a DHAP-Lys imineintermediate, which was trapped by the reduction.

Since the time this mechanism was proposed, considerable refinement has occurred. A more currentversion (from the late 90’s) was proposed by Morris, based on site-directed mutagenesis:


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So far, except for a brief reference to bound metal ions, we have only considered reactions that can becatalyzed by amino acid sidechains. There are only a few types of sidechains, and they can't doeverything, so enzymes enlist the aid of non-protein components, "cofactors".

Vitamins and Minerals

Cofactors: Coenzymes and Prosthetic groupsCofactors are non-protein compounds that aid in catalysis.

Prosthetic groups are tightly-bound cofactors that may sometimes be thought of as part of theenzyme. Prosthetic groups may be organic, inorganic, or organic complexes of inorganic ions.

Coenzymes may often be thought of as cosubstrates. They are organic compounds thatparticipate in the reaction and, in a single turnover, may not be regenerated. Even ATP and its XTPrelatives were once called coenzymes, but are now thought of as substrates. Many of the coenzymescontain AMP:

H2COPO

OO

OHOH

O N

NN

N

NH2

Many cofactors arise from dietary vitamins and minerals. In the partial list below, the vitamin forms


92

shown in ().

Pyridine Nucleotide Coenzymes:NAD+/NADH, Coenzyme 1, Cozymase (from niacin)NADP+/NADPH (from niacin)Structure:

N

NN

N

NH2

H2COPO

O

OHOH

ON

CO

NH2

H

O

H2COPOO

OHOH

O N

NN

N

NH2

OPO

O

O

H2COPOO

OHOH

O

H H

NH2

OC

NO

OHOH

H2C

NAD+ NADH

Class of reaction:2-electron oxidation-reduction reactions (most dehydrogenase reactions)Examples of enzymes

Lactate dehydrogenase, Alcohol dehydrogenase,

Flavin Coenzymes (Vitamin B2, Riboflavin, coenzymes)StructureFMN/FMNH2 (Flavin mononucleotide)

N

N

N

H3C

H3C

O

O

NH

CH2CHOHCHOHCHOHCH2OPO3

-2

N

N

N

H3C

H3C

O

O

NH

CH2CHOHCHOHCHOHCH2OPO3

-2

H

H

FAD (Flavin adenine dinucleotide) (FADH2 form as for FMNH2)


93

N

N

N

H3C

H3C

O

O

NH

CH2CHOHCHOHCHOH

H

H

CH2 OO

OH2COP

OO

O

OHOH

OP N

NN

N

NH2

Class of reaction1-electron transfer reactions2-electron transfer reactions

Many reactions involving O2 involve a flavin and an iron ion. (A one electron reduction leads to asemiquinone form with an unpaired electron.)Examples of enzymes

Succinate dehydrogenase, α-Lipoyl dehydrogenase, glucose oxidase (FAD, 2 e-), NADHdehydrogenase (FMN, 2e-); Flavin:CoQ oxidoreductase.

Note: A typical series of reactions in metabolism follow the sequence:

+ H2O

NADH + H+NAD+

FADH2FAD

R C CH2 R'O

R CH CH2 R'OH

R CH CH R'R CH2 CH2 R'

In the Krebs Cycle, these compounds are succinate, fumarate, malate and oxaloacetate. In fatty acidoxidation, they are fattyacyl groups. In fatty acid synthesis, the reactions are in the opposite order,and the compounds are fattyacyl Acyl-Carrier Protein derivatives and the coenzyme is NADP+. Thereare certainly exceptions; sometimes, flavins participate in oxidations of alcohols to ketones andsometimes pyridine nucleotides participate in desaturations.


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Thiamine Pyrophosphate (thiamine, vitamin B1, "cocarboxylase")Structure

N

N

CH2

NH2

H3C

N S

H

CH2 CH2 O P O PO O

OOO

Class of reactionAcyl transfer reactions, oxidative decarboxylations.

Examples of enzymes: Pyruvate decarboxylase portion of pyruvate dehydrogenase complex., acetoinsynthetase, transketolase

General mechanism

H+N

C SCR

O H

NC SC COH OH

R R'R''

R C CR'

O OH

R''

NC S

NC SCRO

H

HThis intermediate could attacka carbonyl or other electrophile,depending on the specificityof the enzyme

Lipoic AcidStructure - Forms an amide with a protein amino group

CO

NSHSHS S

CO

N

Lipoamide Dihydrolipoamide

Class of reactionAcyl transfer reactions.

Examples of enzymes: Lipoic reductase/transacetylase portion of pyruvate dehydrogenase.


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Coenzyme A (calcium pantothenate)

N

NN

N

NH2

H2COPO

OO

OH

O

POO

O

CH2 CCH3

CH3

CHOH

CO

NH CH2 CH2 CO

NH CH2CH2 SH

OPO

OO

Acyl-carrying group

An acyl carrier (acetate, fatty acids, etc.)

CoA activates an acyl group in two ways:1) Thioesters are "high-energy" compounds (i.e., hydrolysis is exergonic) and R-S- or R-SH is a

good leaving group, so the acyl group is activated for attack by a nucleophile:

+

SHCoA

C CH3

O

elihpoelcuN

Nucleophile

CoA S C CH3

O-

NucleophileCoA S C CH3

O

2) The a protons of the acyl group of acyl CoA is slightly acidic because of resonancestabilization by the carboxyl oxygen and electron withdrawal by the sulfur, so the a carbon and bedeprotonated to act as a nucleophile:

+

acetoacetyl-CoAfree CoA

nucleophilic attack

resonance

CoA S C CH2 C CH3

O OCoA SH

CoA S CO

CH3CoA S C CH2

O-

CoA S CO

CH2CoA S CO

CH2

H

Thissequence appears to occur in the anaerobic soil bacterium Clostridium kluyveri. In fatty acid synthesis


96

in the liver, there's another step in the middle in which the CoA enolate attacks a CO2 and becomescarboxylated. The acyl CoA attacks the second acyl CoA during the subsequent decarboxylation. Allthe principles are the same, there's just an extra step.

BiotinStructure

NHHN

SCH2 CH2 CH2 CH2 C

O

O

O

Forms an amide bond with protein, bound form called biocytin.

Class of reactionCarboxylations

Examples of enzymesPyruvate carboxylase, Acetyl-CoA carboxylase, Propionyl-CoA carboxylase

Mn++ CH2HO

OC H

O

NN

CCOO

O

S

Suggested mechanism for pyruvate carboxylaseStrongly bound by a protein from egg called avidin; avidin inhibits all biotin enzymes.


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Pyridoxal Phosphate (pyrydoxine), vitamin B6

N CH3

O-C

O H

H2COPOO

O

H

Class of reactionDecarboxylation, dehydration of amino acids, sulfur incorporation.

Examples of enzymes: Histidine decarboxylaseGeneral mechanism:

+ H2O

NH3C

HO CH2OPO3-2

H

CNC CHOCH2

O

O

NH3C

HO CH2OPO3-2

CHN

C CCH2HOH O

O

H

NH3C

HO CH2OPO3-2

CO H

ketoacid(Oxidation)

-OH--CO2

-

-HOCH2

(+SH-)

Coenzyme Q, Ubiquinone - an electron carrier

O

OH3CO

CH3H3COCH3

CH2CCHCH2 H6-10

Hemes (not a vitamin)Structure (Iron Protoporphyrin IX, there are others)


98

N

N N

N

CH3

CH3

CH3

CH3

CH

CH

CH2

CH2

CH2

CH2H2C

CH2

C

C

OO

OH

HO

Fe

Class of reaction1-electron oxidation/reduction, disproportionation, multi-electron oxidation/reduction (in

conjunction with other metal ions)Examples of enzymes:

Catalase, peroxidase, cytochrome P-450

Vitamin C (reducing agent)

O OCHOHCH2OH

O O

O O

OH

CHOHCH2OH

HO

Tetrahydrofolate (folic acid)

n

HN

N

N

NH2NH

HCH2 N

H

OCO

NH CHCOO-

CH2 CH2 CO

NH Glutamate

Class of reaction: 1-carbon transfer reactions (various oxidation states)Example: Thymidylate synthetase (methylation of dUMP to dTMP)

Cobalamin (vitamin B12, cyanocobalamin)Structure

Heme-like "corrin" ring; cofactor form has 5'-deoxyadenosine attached)


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Class of reaction:1-carbon (or hydrogen) transfer/isomerization reactionsExamples of enzymes: methylmalonyl mutase, dehydration of ribose nucleoside phosphates inLactobacillus

S-Adenosyl Methionine (Methyl donor)

H2C

OHOH

OSCH3

CH2CH2CHH3NCOO N

NN

N

NH2

(non-heme) Metal IonsZn++

Alcohol dehydrogenase, Class II aldolase, DNA polymerase I, RNA polymerase

S Cys

N N

His

ZnS

H2O

Cys

Mg++

All kinases, many other phosphoryl transfer enzymes and adenylating enzymes, Aminoacyl-tRNA synthetase.

N

NN

N

NH2

H2COPO

OO

OHOH

OPOPO

O

OO

O

Mg++

Fe++/Fe+++

Many oxidoreductases or enzymes that employ transient oxidation/reduction or stabilization ofintermediates, electron transfer proteins (ferredoxin, HIPIP).


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Fe

SS Cys

S

FeSCys

Fe S

FeS

S

S Cys

Cys

FeSCys

SCysFe

S

S

Cys

Cys

SS

Two-iron cluster Four-iron clusterHeme iron is an electron or ligand carrier or redox agent (described above).

Cu+/Cu++

Oxidases, superoxide dismutase, polyphenol oxidase, plant nitrite reductaseRecent X-ray structure of nitrite reductase (strereo pair on the left):

MolybdenumNitrogenase, nitrite reductase. Exists in clusters, but more commonly bound to a cofactor

known as molybdopterin:

HN

N N

N

OO

H2N POR

O-O

SMoSO

H

H

or

HNN

NN

O

O

NH2

PRO O-

O

S

S

O

HH

NHN

N N

O

O

H2N

PORO-

O

S MoS

O

H H

(more info at http://metallo.scripps.edu/PROMISE/MOCOMAIN.html)


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Naming EnzymesTrivial names

common namesgenerally, "substratease"this pattern is especially common for hydrolytic enzymes

amylose - amylaseprotein - protease or proteinaselactose - lactase

for other enzymes, often the name of the reaction is identifiedsubstrate - oxidase

NADH oxidasesubstrate - dehydrogenase

lactate dehydrogenasesubstrate - isomerase

triosephosphate isomeraseSometimes, there are "very" trivial names that give no real information about the reactions they

catalyze:Ficin, Papain, Bromelain, Trypsin, Subtilisin, Pepsin and Chymotrypsin are all proteases. How

would you know what they do?

Systematic namesThese are recommended by the Enzyme Commission (see below).They identify the nature of the reaction and the reactant and usually the product.

The best system is a numbering system invented by the Enzyme Commission.:The Enzyme Commission Nomenclature system(a subcommittee of the International Union of Biochemistry)

The Enzyme Commission system consists of a numerical classification hierarchy of the form"EC i.j.k.l", in which "i" represents the class of reaction catalyzed (see classes below), "j" denotes thesub-class, "k" denotes the sub-subclass, and "l" is usually the serial number of the enzyme within itssub-subclass. The criteria used to assign "j" and "k" depend on the class and represent details useful todistinguish one activity from another. The Enzyme Commission's report gives a list of guidelines to aidin assigning an enzyme to its proper category. In addition, a systematic name with a logical form(defined for each class of reaction) is given together with a rational common name. All enzymes areplaced into one of the following classes:

1. Oxidoreductases2. Transferases3. Hydrolases4. Lyases5. Isomerases6. Ligases

The Classifications:1. Oxidoreductases catalyze oxidation-reduction reactions. Their systematic names have the

form "donor:acceptor oxidoreductase", where the donor is the molecule becoming oxidized (donating ahydrogen or electron). Their recommended common names have the form "donor dehydrogenase",unless O2 is the acceptor, in which case "donor oxidase" is permitted. The subclass describes thechemical group on the donor that actually becomes oxidized (e.g., an alcohol, keto- or aldo-group). Sub-


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subclasses generally, but not always, distinguish among acceptors (e.g., NAD(P)H, cytochromes, O2,etc.).

2. Transferases catalyze group transfers from one molecule to another. Systematic nameslogically have the form "donor: acceptor group transferase". Recommended common names are "donorgrouptransferase" or "acceptor grouptransferase", but "acceptorkinase" (e.g., hexokinase) is used formany phopshotransferases. The subclasses distinguish among the various groups that are transferred isa general way (e.g., one carbon transfers, acyl transfers, glycosyl transfers) and the sub-subclassesemploy greater detail in distinguishing among the groups transferred. It is noteworthy thattransamination reactions between an amine and a ketone are classified as group transfer reactions eventhough the ketone becomes reduced to an amine and the amine becomes oxidized to a ketone.

3. Hydrolases catalyze hydrolytic cleavage of C-C, C-N, C-O or O-P bonds. They areessentially group transfer reactions but the acceptor is always water. For this reason, and probablybecause of the ubiquity and importance of hydrolases, they are awarded their own class. Since thereactions are comparatively simple (two substrates, one of which is always water), the systematicnames are also simple: "substratehydrolase". Common names may simply be "substratease". However,since a substrate may have more than one hydrolytically labile bond, it is useful to include the kind ofgroup being transferred to water (e.g., methylesterase, O-glycosidases). The subclass number reflectsthe need to specify the bond being hydrolyzed and the sub-subclass further defines the nature of thesubstrate. In the case of proteases (peptidyl-peptide hydrolases), the sub-subclass reflects propertiesof the enzyme itself (e.g., metalloproteases, serine proteases, etc.), rather than the substrate.

4. Lyases catalyze elimination reactions resulting in the cleavage of C-C, C-O, C-N or a fewother bonds, or the addition that constitutes the reverse of these reactions. The systematic names arewritten as "substrate group-lyase", in which the hyphen is not optional. If the reverse (addition)reaction is more important than the elimination reaction, the name "product synthase" may be used.Subclasses contain enzymes that break different bonds (C-C, C-N, etc.), and sub-subclasses distinguishamong enzymes on the basis of the identity of the group eliminated.

5. Isomerases catalyze structural rearrangements. Their recommended names correspond to thekind of isomerizations carried out by members of the different subclasses: racemases and epimerases,cis-trans-isomerases, tautomerases, mutases and cyclo-isomerases. The sub-subclasses depend uponthe nature of the substrate.

6. Ligases catalyze bond formation coupled with the hydrolysis of a high-energy phosphatebond. The systematic names are written "A:B ligase", and may specify "ADP-forming", etc.,depending on the coupled energy source. Common names often include the term synthetase, which theCommission discourages because of confusion with the name synthase (which does not involve ATPhydrolysis). Subclasses are created on the basis of the kind of bond formed (C-C, C-O, etc.); sub-subclasses exist only for C-N ligases.

Examples of subgroups for Oxidoreductases:EC i.j.k.l

1. Oxidoreductases1.1 acting on CH-OH groups

1.1.1 NAD+ or NADP+ as acceptor1.1.2 Cytochrome as acceptor1.1.3 Oxygen as acceptor1.1.99 other acceptor

1.2 acting on aldehyde or oxo groups1.2.1 NAD+ or NADP+ as acceptor


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1.2.2 Cytochrome as acceptor1.2.3 Oxygen as acceptor1.2.4 Disulfide groups as acceptors1.2.7 Iron-sulfur protein as acceptor1.2.99 other acceptor

1.3 acting on CH-CH groups1.3.1 NAD+ or NADP+ as acceptor1.3.2 Cytochrome as acceptor1.3.3 Oxygen as acceptor1.3.7 Iron-sulfur protein as acceptor1.3.99 other acceptor

1.4 acting on CH-NH2 groups1.4.1 NAD+ or NADP+ as acceptor1.4.2 Cytochrome as acceptor1.4.3 Oxygen as acceptor1.4.4 Disulfide groups as acceptors1.4.7 Iron-sulfur protein as acceptor1.14.99 other acceptor

1.5 acting on CH-NH groups1.5.1 NAD+ or NADP+ as acceptor1.5.3 Oxygen as acceptor1.1.99 other acceptor

...and so forth.

To summarize the EC system

1. Oxidoreducatses EC 1.j.k.l"donor:acceptor oxidoreductase"

e.g.,ethanol:NAD+ oxidoreductase" is alcohol dehydrogenaseEC 1.1.1.1 (see the table passed out in class)i = 1. -> oxidoreductasej = 1. -> CH2OH is being oxidizedk = 1. -> NAD+ is acceptorl = 1. -> the donor is ethanol

orformate:NADP+ oxidoreductase" is "formate dehydrogenase (NADP+)EC 1.2.1.43 (see the table passed out in class)i = 1. -> oxidoreductasej = 2. -> aldehyde or carboxyl (in this case COOH) is being oxidizedk = 1. -> NADP+ is acceptorl = 43. -> the donor is formate

2. TransferasesAX + B -> A + BXX can not be H: or H. because this would be an oxidoreductaseB can not be H2O because this would be a hydrolase


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IF AX is ATP, the enzyme is a kinase, which is a transferase"donor grouptransferase" or "acceptor grouptransferase", "kinase" is used for phosphoryl

transfer enzymese.g.,

EC 2.3.1.9 is Acetyl CoA acetyltransferase2 AcCoA -> Acetoacetyl CoA + CoASHi = 2. -> transferasej = 3. -> an acyl group is transferredk = 1. -> an acyl group, not aminoacyl group is transferredl = 9. -> acetyl CoA is the acceptor

3. HydrolasesAX + H2O -> AH + XOH"substrate hydrolase" or "substrate grouphydrolase"

e.g.,EC 3.2.1.1 is 1,4-a-D-Glucan glucohydrolase (trivial name a-amylase)i = 3. -> hydrolasej = 2. -> acts on glycosyl compoundsk = 1. -> hydrolyzes O-glycosyl bondsl = 1. -> produces a glucan rather than glucose or maltose

4. LyasesNon-hydrolytic breakage of bonds, usually involving C-C, C-N, C-S, etc.Includes decarboxylases (= carboxy-lyases), and additions to double bonds

R C C R'H H

HX R C C R'H X

H H+

or the reverse (addition) of this reaction. XH can be water in addition/elimination reactions."substrate group-lyase" or "product synthase" (not synthetase) if the addition reaction is most

important.EC 4.1.1.53 is phenylalanine carboxy-lyase (a decarboxylase)i = 4. -> lyasej = 1. -> acts on C-C bondsk = 1. -> acts on carboxyl groupsl = 53. -> specific to this enzyme

5. Isomerasestrivial name depends on the kind of isomerizationEC 5.1.3.20 is D-Ribose ketol isomerase (ribose isomerase)i = 4. -> isomerasej = 1. -> interchange keto and alcohol groupk = 1. -> denotes substratesl = 53. -> specific to this enzyme

6. Ligases


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A + B + ATP AB + AMP + PPi

ADP + Pi

or

"A:B ligase", may designate AMP forming (example above)May be called "synthetase".EC 6.2.1.3 is Acetate:CoA ligase (Acyl-CoA synthetase)i = 6. -> ligasej = 2. -> forms C-S bondsk = 1. -> acid-thiol bond formedl = 3. -> specific to this enzyme

Examples of enzymes from some of these classes:HydrolasesOxidoreductasesLigases

Specific groups of enzymes

Selected for further emphasis: the hydrolases and the oxidoreductases.

Hydrolases

A-X + H20 AH + XOH

A. Types of enzymes belonging to the hydrolases

EC 3. . . indicate hydrolases. The second number indicates the type of hydrolase (11types).

No. Type Substrate No. of enzymes

EC 3.1._._ esterases R-C-O-R' 62

simple esters:R-COO-CH3

triglycerides (lipases)

phosphatases

O-P-O-RO

O

--


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sulfatases

O-S-O-RO

O-

etc.EC 3.2._._ Glycosidases

O

O

OCH2OH CH2OH

HOOH

OH

OH

OHO

EC 3.3._._ splits ether 45bonds R- CH2-O-CH2-R'

EC 3.4._._ proteases peptide bonds 39(Now EC 3.11._._)

EC 3.5._._ splits non-peptide 46bonds to N, e.g.,

H2N C NH2

O

EC 3.6._._ ˆEC 3.7._._ ÍEC 3.8._._ ˝ 18EC .3.9._._ ÍEC 3.10._._ ¯ ____

Total 149

B. A question of one aspect of mechanism of action of a hydrolase is the position of splitting ofthe bond with water.

21

CO

R O R'

The mechanism of action is quite different depending on which side of the 0 the bond is split. Usingesters as examples:


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OH H

R'OROC

-

OH H

1

CO

R O R'

+R CO

OH OH-

R'

OH H

CO

R O R'

2

Nucleophilic Electrophilic

In order to determine where splitting occurs use H180H. 180 is not radioactive. Its location can bedetected after separation of the products by mass spectrometry. The question is where does the wateroxygen go?The possibilities are:

+R C

O

OHHOR'

18 and

+R COH

O18

H OR'

1. In the case of the esterases:

HOR'R CO

OH+

18

So splitting occurs between C-0.

2. In proteases:

R CO

OH+

18H2NR'

(Duh!)

3. In glycosidases:

+O

CH2OH

OH

OHOHO

OCH2OH

HOOH

OHO H18

C. Concept of enzyme specificity using glycosidases as examples.Glycosidases have varying degrees of specificity. We'll use a group of hydrolases to talk about

how you can determine the specificity of an enzyme. However, the general concepts are appropriatefor determining the specificity of any enzyme.

1. Specificity must be determined using substrates, not using competitive inhibitors.

2. Specificity involves two factors.a. The ability to recognize a compound and bind it.


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b. The ability to convert a substrate to product.

The measure of specificity is therefore Vmax/KM for different compounds. The higher the ratio ofVmax/KM, the higher the specificity. In other words, what you want to have Vmax as high as possibleand Km as low as possible.

c. To determine specificity for a substrate.1) determine Vmax and KM by the Lineweaver-Burk method, or

2) measure Vo and [S]o << KM, where Vo = Vmax KM

[S]o

3. Examples of some ideas on the specificity of the glycosidases.

Our general substrate will be maltose (glucosyl α-l,4-glucose).

OCH2OH

HOOH

OHO

OH

OH

CH2OHO

OH

By making systematic changes on the substrate we can get an idea of specificity.

In general, the glycosidases split the compound between C1 and 0 rather than between 0-C4. There isone important (semi) exception to this. Sucrose is a non-reducing disaccharide. The glycosidic bondinvolves sharing an oxygen by C1 of the glucosyl moiety and C2 of the fructosyl moiety. β-Fructofuranosidase replaces the oxygen attached to the fructose, whereas an a-glucosidase replaces theO attached to the glucose.How to tell? - By using H180H we can distinguish between the two types of sucrases (invertases).What is the difference in the products formed by the two sucrases in H2O?

Reducing sugars (di or oligosaccharides) don't possess this ambiguity because the two units aredifferent - only one (the "glycone") has its reducing end engaged in the bond; the other (the "aglycone")does not.The two monosaccharides in the disaccharides are referred to as the glycone and aglycone as follows.

OCH2OH

HOOH

OHO

OH

OH

CH2OHO

OH

Glycone Aglyconea. What is the importance of the aglycone in specificity? Test it by keeping the glycone constantand varying the nature of the aglycone and measuring Vmax/Km

Some data for β-galactosidase are:Substrates: galactosyl-R where R = aglycone


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Aglycone (R) KM Vmax Vmax/KM(mM) (mol/min•mg)

1) glucose (lactose) 1.9 6.5 3.52)-Oφ 1.47 10.4 7.1

3)-O-p-nitroφ 0.0531 22.4 420

4)-O-o-nitroφ 0.161 178 1100.

5)-OCH3 split but I don't have the data

Conclusions

1) A change in the aglycone does not prevent the enzyme from splitting the glycosidic bond.

2) The nature of the aglycone does have an effect on relative specificity

What about the nature of the glycone?In general, the nature of the glycone is absolutely essential to have activity with a given enzyme.

b. Specific nature of the glycone

1) glucosidases are specific for glucose as the glycone

2) galactosidases are specific for galactose as the glycone. The specific glycone is all importantin determining whether one type of enzyme will split it or not.

c. Stereochemistry: importance of the α- or β-glycosidic bond

OCH2OH

HOOH

OHO

OH

OH

CH2OHO

OH

Maltose (glucosyl a-l,4-glucose) is an α-glucoside,maltase is an α-glucosidase

OCH2OH

HOOH

OH

O OHO

CH2OH

OH

OH


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Cellobiose (glucose -l ,4-glucose) is a β-glucoside,cellobiase is a β-glucosidase.

Holds true for higher polymers also.Starch is α-l,4-glucose polymer amylasesCellulose is β-l ,4-glucose polymer cellulases

d. D- or L-configuration

O

OH

OHHO

CH2OH

OR

O

OH

OHHO

CH2OH

OR

a-D-glucose a-L-glucose

The D-glucose is the form normally found in nature. Therefore,the enzymes have evolved to work on these and only the D-formis split. L-form often binds but k2 = 0.

e. Size of ringMany sugars can exist in either the furanose or pyranose structures. Which one is favored

depends on bond angle strain and steric interactions. The difference in covalent structure depends onwhich oxygen atom attacks the carbonyl group to form the hemiacetal bond -or- on where the carbonylcarbon is.

O

OH

OHHO

CH2OH

OR

O

OH

OH

CH2CH2 OHHO

pyranose structure furanose structureFructose exists as a mixture of pyranose and furanose in solution.

It has been found that all enzymes which work on 6-C aldo sugars require the pyranose form,while all those which work on 6-C keto sugars (e.g., fructose) require the furanose form.

f. Attachment of the aglycone to the glyconeGlycosidic bonds are acetal bonds. Only attachments between the reducing (anomeric) carbon

of the glycone and a hydroxyl group of the aglycone are acetals. For instance, attachment to C2, C3,C4 or C6 of glucopyranose would produce ethers, which cannot be cleaved by glycosidases. C1 ofaldoses and C2 of ketoses are therefore unique because only they can be involved in "glycosidic"bonds.

g. Attachment of the glycone to the aglycone


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If the aglycone is a hexopyranose, the glycone can be attached at positions C1, C2, C3, C4, or C6. Allform glycosidic bonds of 1:1, 1:2, 1:3, 1:4, 1:6. All of these types are found in nature. There is aseparate type of enzyme to split each type of bond.

h. Conformation of the glyconeRemember that pyranoses can exist in chair and boat forms. The relative stabilities (and

therefore, populations) of the chair and boat forma are determined largely by steric interactions amongring substituents.

O

OHOH

HO

HO

CH2OHOOH

OH

OH

HO

CH2OH

Chair Boat

Enzymes are thought to have a preference for the chair configuration. An example is the enzymelysozyme. It is specific for the chair form, converting it to the half-chair form on binding. This straininduced in the substrate is partially responsible for the ability to split the glycosidic bond.

1. What is the effect of substrate size?

Maltase only acts on the substrate maltose, which is a disaccharide. Amylase, on the other hand,requires a substrate that is a polymer larger than maltotriose (glc α-l ,4-glc α-l ,4-glc).

For some enzymes the size specificity is relative, not absolute. For example:

PolygalacturonaseSubstrate Relative RateDigalacturonic acid 0.0Tri- 1.7Tetra- 2.2Penta- 12.8Hexa- 15.8DP=12 52.0DP>100 100.0

CellulaseSubstrate Relative RateCellobiose 1.2Cellotriose 46.0Cellotetrose 79.0Cellopentose 100.0Cellohexose 96

j. Where the enzyme splits the polymer


112

Polysaccharides have a reducing end, a nonreducing end, and many monomer units in between.If they all are attached by the same kind of bonds, have the same stereochemistry, conformation, etc.,are all bonds cleaved at equal rates?

Of course not!.The majority of the exoglycosidases direct action to the non-reducing end.Endoglycosidases cleave only interior glycosidic bonds and have very low rates for small

fragments.

k. Sensitivity to changes in the bridging atom in the glycosideNot all glycosides have bridging oxygen atoms. Sulfur and nitrogen bridges also exist.•Thioglycosides are important flavor precursor in smelly leafy vegetables like mustard and

cabbage. Thioglycosidases are important in flavor development.•Some important N-linked glycosides have another name - nucleosides (nucleotides). These

include purine and pyrimidine attachments to ribose in nucleic acids, B-vitamins and nucleosidetriphosphates. The "aglycone" in these compounds is not a sugar.

There is essentially no cross reactivity among the enzymes specific for these three groups.

1. Sensitivity toward modification of the 2, 3, 4 or 6 position of glycone.The enzymes are specific for configuration of these -OH.Can't modify or delete the 2, 3, 4 -OH without loss of activity.(Sometimes can leave off the 6-OH)Different enzymes are specific for different structures.As luck would have it, the configuration of these OH groups is used as the basis for the naming

system for sugars, so chemists and enzymes recognize the same structural features of the substrates.

M. Modification of the heterocyclic 0Via synthetic chemistry, the ring O can be replaced with S or CH2. In either case, there is no

longer a glycosidic bond and none of the glycosidases can split the molecules.

Summary: Specificity involves two things:1. The ability of the compound to bind into the active site.

2. The ability of the enzyme to convert the compound into P.

Focus on some classes of hydrolasesThe AmylasesThese have become the single most important or useful group of enzymes commercially in the pastfive years.

The amylases are used to produce some 1011 pounds of fructose per year and even larger amounts ofglucose.

a-amylase glucoamylase glucoisomerase

Starch

dextrins

glucose

fructose


113

14 tons of enzymes are involved in the process/year.

Starch is an a-1,4 polymer primarily. The amylose fraction is not branched and has only a-l,4 linkages.The amylopectin has some a-1,6 branching.

The types of amylases are:1. a-amylases split only α-l,4 glucosidic linkages2. b-amylases3. glucoamylases - split α-l,4 glucosidic linkages rapidly, α-1,6 more slowly.4. pullulanases - split α-l,6 linkages, the so-called debranching enzymes

When the amylases act upon starch, the following changes occur:

1. Decrease in viscosity due to the hydrolysis of starch into smaller fragments. Very sensitivemethod for detection of amylase activity..

2. Decrease in I2 color. Starch (amylose portion) stains a deep blue color with I2, because theiodine forms a complex in which it is encased in the helical structure of the starch As the starch ishydrolyzed the color goes from blue to brown and then colorless. The decrease in blue color (boundiodine) can be used as very sensitive method for following starch degradation.

3. Reducing groups are formed. These can be measured by use of 3,5-dinitrosalicylic acid. Onheating, the reagent turns red in the presence of reducing groups. The intensity of color is proportionalto the number of reducing groups.

4. Measure maltose and/or glucose formed during the hydrolysis.a) To determine maltose concentration:After the amylase treatment, add maltase to an aliquot of the digest, then measure the increase

in reducing groups formed:Digest: Starch + H20 --> dextrins + maltose (measure reducing groups), then

maltose + maltase --> 2 glucose (measure reducing groups again)b) To measure glucose specifically, use the Statzyme® reagent, which uses glucose oxidase and

peroxidase plus a chromogen:

glucose + H20 + O2 --> gluconic acid + H202 , in the presence of glucose oxidase, then measurethe peroxide produced by using peroxidase

H202 + chromogen --> brown color

5. Use p-nitrophenyl glycosides (e.g., p-nitrophenyl trimaltoside) to obtain colored products

How to distinguish among the different amylases.

It is easy enough to distinguish pullulanase from a-amylase, b-amylase and glucoamylase since it onlyhydrolyzes a-l,6 linkages.It would show no activity on amylose, would debranch amylopectin.

How about the difference among α−, β− and glucoamylases?


114

Criterion α-amylase β-amylase glucoamylase1. Source all living higher plants microorganisms

organisms a very few bacteria

2. Mode of attack endo-splitting, exo-splitting exo-splitting random (non-reducing (non-reducing

end) end)3. Δη/Δreducing fast slow slow

groups

4. ΔI2/Δreducing fast slow slowgroups

5. Rate of reducing group formation - depends on [E] -6. Rate of maltose liberation/

reducing groupsvery little 1 0

7. Rate of glucose liberation/ # reducing groups found

essentially zero 0 1

8. Inhibited by -SH reagents No + No

9. Configuration of productα β β

Pectin splitting enzymesPectic enzymes are very important in food systems because they function during

ripening/maturation of plant tissues. They are useful in clarification of juices, etc.

There are three types:1. Those which split the ester bond in pectin to form pectic acid + methanol.

+H2O

CH3OHOO O

O

OH

OH

C OCH3O

O

OH

OH

C OCH3O

O

OH

OH

CO O-

O

OH

OH

C OCH3O

OO O

These belong to the esterase class.

a. Experimentally determine by measuring H+ liberation.c. Or, measure methanol production.


115

b. Little change in viscosity unless Ca2+ added.

2. Polygalacturonasesa) These split the glycosidic bond using H20:

O

OH

OH

CO X

O

OH

OH

CO X

OO O OO

O

OH

OH

CO

HO

X

O

OH

OH

CO

OH

XH2O

b) Determine activity by:1) reducing groups formed2) viscosity change

c) There are 4 types of polygalacturonases.endopolymethylgalacturonase (pectin as substrate)exopolymethylgalacturonase (pectin as substrate)endopolygalacturonase (pectic acid as substrate)exopolygalacturonase (pectic acid as substrate)

3. Pectate lyasesThese split the glycosidic bond by removal of H.

+O

OH

OH

CO

OH

X

O

OH

OH

CO

H

X

O OOO OO

OH

OH

CO X

O

OH

OH

CO X

There are 4 types of pectate lyases.a. endopolymethylgalacturonic acid lyase (pectin as substrate)b. endopolygalacturonic acid lyase (pectic acid as substrate)c. exopolymethylgalacturonic acid lyase (pectin as substrate)d. exopolygalacturonic acid lyase (pectic acid as substrate)

Can distinguish the polygalacturonases from the pectate lyases.

1. For each bond split by lyase get one reducing group and one double bond which has Amax at 235nm.

2. With polygalacturonases no double bonds formed, only reducing groups.


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3. Lyases found only in microorganisms; polygalacturonases found in both higher plants andmicroorganisms.

Animals do not contain any pectic enzymes.

Proteolytic Enzymes

The proteolytic enzymes all have in common the ability to catalyze the hydrolysis of peptide bonds.Their role in vivo is the hydrolysis of proteins and peptides. Many proteases also hydrolyze esterbonds.

Y NHCHCNCCX

R

R'O

OH

H

Different types of proteolytic enzymes can be distinguished on the basis of substrate specificity.

A. Nature of the R group.

1. For trypsin and trypsin-like enzymes (of which there are many), R is the side chain residue ofLys or Arg. The enzyme requires a positive charge on the group adjacent to the peptide bond to besplit (the one that furnishes the carboxyl group to the peptide bond).

2. For chymotrypsin and chymotrypsin-like enzymes, the highest rate of hydrolysis is obtainedwhen R is a Phe, Tyr or Trp residue, although some aliphatic, uncharged sidechains are also accepted..

3. For papain, R can be essentially any amino acid residue provided the Y residue, (the residuetoward the N-terminal end) is Phe, Tyr, or Trp. In other words, the major specificity factor for papainis one residue removed from the peptide bond that is split.

B. Nature of the R' group.

For trypsin, chymotrypsin, papain, etc., the nature of the R' group is of relatively less importance indetermining specificity, but the rate of hydrolysis of the peptide bond is somewhat dependent uponthe amino acid residue that furnishes the HN-group of peptide bond.

However, the following amino acid derivatives are hydrolyzed, provided that the R groups areconsistent with the specificity of the enzyme.

1. Simple amides

R CO

NH22. Hydroxamides

R C NOHO H


117

3. Aromatic amides (or esters - remember the chromogenic substrates used for trypsin andchymotrypsin in lab?)

XR C

ONH

4. Esters

R CO

O (CH2)nCH35. Thioesters

R CO

S(CH2)nCH3

The ester substrates are split more rapidly than the amide substrates, not because of better fit at theactive site, but because the Ea for ester hydrolysis is 6 kcal/mole while it is ~12 kcal/mole for amide.Again, it should be stressed that the specificity for R must be supplied in order for the compound toserve as substrate.

C. Configuration of amino acids

For all of the commonly occurring proteases, the configuration must be L. With D-amino acids there isno hydrolysis, although binding may occur (inhibition!).

D. Nature of x

1. Endoproteases work best if x is another amino acid (or some blocking agent).2. With carboxypeptidases, x must be -OH so that there is a free C0O-.

E. Nature of y

1. Endoproteases work best if y is another amino acid or at least is an acyl group (acetyl,benzoyl...)

2. With amino peptidase the y must be H so that the -NH2 is free.

F. Size of the substrate

1. With dipeptidases substrate can't be larger than the dipeptide; x and y must be OH andH, respectively.

2. With tripeptidases 3 amino acid residues are required.3. For the endoproteases, the carboxypeptidases and

aminopeptidases, size is relatively unimportant provided the other specificityrequirements are met.

For example: N-acetyl-L-tyrosine methyl ester


118

HO CH2 C C OCH3

ONHC

CH3 O

is an excellent substrate for the endoprotease ChTr.It has been shown that other amino acid residues added at HN- will increase the specificity by

factor of 2-5; however, this increase is due to better binding of the substrate.

The acid proteases such as pepsin, chymosin, etc., are a little more fussy with respect to thesize of substrate and require that an amino acid furnish the HN group. The acid proteases will nothydrolyze ester substrates. The binding site appears to be much larger than the active site of ChTr ortrypsin. For example, the E. parasitica protease hydrolyzes Leu-Leu NH2 more slowly than Leu-Leu-Leu NH2 and hydrolyzes larger peptides even faster. Additional residues have an effect and therefore,must interact with the enzyme.

G. The proteases can be divided into 4 groups based on the type of functional groups in the activesite.

1. Serine proteases - These contain essential Ser-0H, imidazole of histidine and a COO- ofAsp.

Examples: trypsin, chymotrypsin, thrombin, subtilisin

2. Sulfhydryl proteases - These contain essential Cys-SH, imidazole histidine and The b-carboxyl group or amide of Asp or Asn

Mechanism appears to be very similar to that of the serine proteases.Example: Papain, ficin...

3. Carboxyl (acid) proteases - Contain two C0OH groups furnished by Asp. pepsin,chymosin, Mucor pusillus protease, Endothia parsitica proteases

4. Metalloproteases - In addition to amino acid residues, these require a metal ion, generally Zn2+

but in a few cases, Co2+.The great majority of the metalloproteases are exoproteases,carboxypeptidases, aminopeptidases, di - and tri -peptidases.

There are a few microbial endoproteases which are metalloproteases.

THE OXIDOREDUCTASES EC 1._._._

As the name indicates, these enzymes carry out oxidation-reduction reactions on the substrate.

They can do this by one or more of the following mechanisms.


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1. Remove H (not H+) from the substrate.2. Remove electrons from the substrate.3. Add 02 to the substrate.

There are 8 general types of oxidoreductases.

reaction acceptor # enzymes ingroup

examples

A. AH2 + B --> A + BH2 NAD+ , NADP+ 132 alcohol dehydrogenaselactate dehydrogenase

B. AH2 + 02 --> A + H202 02 11 Glucose oxidase

C. 2AH2 + O2 --> 2A + 2H20 02 6 polyphenol oxidase

D. A + B + H20 --> A0 + BH2 NAD+, NADP+,02

28 xanthine oxidase

E. A +H20 --> A0 + H20 H202,(A=guaiacol, etc.)

8 catalase, peroxidase

F. A + 02 --> A02 02 13 lipoxygenase

G. A + BH2 + 02 --> A0 + B + H20 O2*NADPH,NADH, others

14 hydroxylation activity ofpolyphenol oxidase

H. Miscellaneous 9TOTAL 221

* note that these are electron donors; O2 is the acceptor. Enzymes that employ pyridine nucleotidecoenzymes to supply additional electrons to reduce oxygen are called "mixed-function" oxidases oroxygenases (depending on whether or not oxygen is incorporated into the product).

Note the following:•Only reactions in A, D, and E would proceed under anaerobic conditions. The others require

oxygen.•In A, B and C, the oxidized form of the product does not contain an additional oxygen atom. In

D-G, the substrate A acquires one or two new oxygen atoms during the reaction. (How could youmeasure this?)

•Even though A, B and C do not involve incorporation of oxygen into the product, B and C stillrequire oxygen and would not proceed anaerobically.

•B and C can be distinguished because B produced H2O2 but C produces water.•D-G involve incorporation of oxygen in the product, but

-in D, the O comes from H2O and appears in AO-in E, the O comes from H2O2 and appears in AO and H2O-in F, the O comes from O2 and appears only in AO2-in G, the O comes from O2 and appears in AO and H2O.

How could you tell?

A. Pyridine Nucleotide-linked dehydrogenases have already been discussed....peroxidases and lipoxygenases are important in food science, but they will be omitted due to lack oftime...


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Polyphenoloxidase, o-diphenol:oxygen oxidoreductase EC 1.10.3.1This is a complicated enzyme with more than one activity. It therefore also has more than one

name: tyrosinase, phenolase, cresolase, catecholase, catechol oxidase. It was discovered in 1856 (!) bySchönbein and is responsible for enzymatic browning in many foods and food products.

PPO may have two types of activity:monophenolase activity

+ H2O

+

OO

X

OHOH

OHOH

X

+ 1/2 O2 +

OH

diphenolase activity.

+ 2H2O22

OO

OHOH

+ O2

Catechol o-benzoquinone

Measurement of monophenolase activity•absorbance at 280 nm•O2 uptake (oxygen electrode)•absorbance change at 470 nm

Measurement of diphenolase activity•absorbance at 470 nm•O2 uptake

a secondary reaction occurs in which the o-benzoquinone forms melanin, using four atoms of O2.

So, the reaction looks complicated. If you began with only a monophenol, you would initially haveonly slow oxidation of the monophenol (to produce some diphenol). The presence of diphenolenhances the rate of monophenolase activity and enables benzoquinone production. The kinetics mightlook like this:


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[P]

time

no diphenol added

diphenol added

Another peculiarity is that the enzyme commits suicide during the reaction, so the reaction comes to ahalt - not because it has reached completion, but because the enzyme is dead. Adding more substratedoes nothing; adding more enzyme initiates the reaction again:

A470 nm

time

additional enzyme added

(~2 min)

It is believed that the irreversible inhibition occurs because of a free radical intermediate that is formedthat destroys the enzyme.

Control of PPO activity in foods1. Ascorbic acid - a reducing agent2. NaHSO3 or SO2 gas - another reducing agent

these were initially thought to reverse the reaction by reducing the products; they arenow known to work by inhibiting the enzyme.

3. Citric acid4. Tiethyldithiocarbamate5. Suicide reagents ("mechanism-based inhibitors")

hit imidazole groups in active sitecondensation with ε-amino groups of lysine


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Current thoughts on mechanism1. resting form of the enzyme is "met-tyrosinase" (I) which contains a Cu++ at the active site.

This form is inactive.2. The diphenol (AH2) reduces the copper to Cu+ to produce "deoxytyrosinase" (II)3. This form binds O2 to yield "oxytyrosinase" (III).4. oxytyrosinase binds

a) a monophenol to give IV, then V to form an o-diphenol intermediate, orb) a diphenol combines with a diphenol to give VI, and is oxidized to an o-

benzoquinone.

Documents

123 Notes, part 3 Enzyme mechanism - Examples of elucidationfst123.fst.ucdavis.edu/~garysmith/123Data.dir/Notes.dir/123_pt3.pdf · Here are some examples of the kind of information