Lecture6enzy advance

The Behavior The Behavior of Proteins: of Proteins: EnzymesEnzymes

Enzyme CatalysisEnzyme Catalysis• EnzymeEnzyme: a biological catalyst

• with the exception of some RNAs that catalyze their own splicing (Chapter 8), all enzymes are proteins

• some enzymes are so specific that they catalyze the reaction of only one stereoisomer, others catalyze a family of similar reactions

• Gibbs free energy (G) Gibbs free energy (G) the relationship between entropy (S) and enthalpy (H), where

G = H - TS

Enzyme CatalysisEnzyme Catalysis• For a reaction taking place at constant tempera

ture and pressure, e.g., in the body

the change in free energy is

• The change in free energy is related to the equilibrium constant, Keq, for the reaction by

G° = H° - TS°

G° = RT ln Keq

Activation Energy ProfileActivation Energy Profile

Progress of reaction

reactants

products

Transition state

Activationenergy

Free energy change

Activation Energy ProfileActivation Energy Profile• an enzyme alters the rate (kinetics) of a reaction, but

not its free energy change (thermodynamics) or position of equilibrium

Progress of reaction

reactants

products

Uncatalyzed reaction

Enzyme-catalyzedreaction

Enzyme CatalysisEnzyme Catalysis• Consider the reaction

H2O2 H2O + O2

No catalyst

Platinum surface

Catalase

75.2 18.0

48.9 11.7

23.0 5.5

Activation energykJ/mol kcal/mol

Relativerate

4 x 1010

1 x 1021

Enzyme KineticsEnzyme Kinetics• For the reaction

• the rate of reaction is given by

• where k is a proportionality constant called the specific rate constantspecific rate constant

• Order of reactionOrder of reaction: the sum of the exponents in the : the sum of the exponents in the rate equationrate equation

A + B P

Rate = [A]t

_ _= =

Rate = k[A]f[B]g

Enzyme CatalysisEnzyme Catalysis• In an enzyme-catalyzed reaction

• substrate, Ssubstrate, S: the molecule(s) undergoing reaction

• active siteactive site: the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions

Enzyme CatalysisEnzyme Catalysis• Two models have been developed to describe

formation of the enzyme-substrate complex• lock-and-key modellock-and-key model: substrate binds to that portion

of the enzyme with a complementary shape• induced fit modelinduced fit model: binding of the substrate induces a

change in the conformation of the enzyme that results in a complementary fit

Also, H2O molecule play a much important role.

Enzyme CatalysisEnzyme Catalysis• Chymotrypsin - catalyzes selective hydrolysis

of peptide bonds where the carboxyl is contributed by Phe and Tyr• it also catalyzes hydrolysis of the ester bond of p-nit

rophenylacetate

O2N OCCH3

chymo-trypsin

O2N O- CH3CO-+

pH > 7

Op-Nitrophenylacetate

p-Nitrophenolate

ChymotrypsinChymotrypsin

Concentration of p-nitrophenylacetate (S)

Reacti

maximum velocity

ATCaseATCase• Aspartate transcarbamylase (ATCase) catalyze

s this reaction

H2N-C-O-P-O-

CH-CO2-

H2N-C-NH-CH-CO2-

+H3PO42-

ATCase

Carbamoylphosphate

Aspartate

N-Carbamoylaspartate

ATCaseATCase

Concentration of aspartate (S)

) maximum velocity

Note sigmoidal shape,which, as we will see, is one characteristic of allosteric enzymes

Enzyme KineticsEnzyme Kinetics• Initial rate of an enzyme-catalyzed reaction

versus substrate concentration

Please see Fig. 5.6 (p156)

Michaelis-Menten ModelMichaelis-Menten Model• for an enzyme-catalyzed reaction

• the rates of formation and breakdown of ES are given by these equations

• at the steady state

E + S ES Pk1

k1[E][S] = k-1[ES] + k2[ES]

rate of formation of ES = k1[E][S]

rate of breakdown of ES = k-1[ES] + k2[ES]

Michaelis-Menten ModelMichaelis-Menten Model• when the steady state is reached, the concentration

of free enzyme is the total less that bound in ES

• substituting for the concentration of free enzyme and collecting all rate constants in one term gives

• where KM is called the Michaelis constant

[E] = [E]T - [ES]

([E]T - [ES]) [S]

[ES] k-1 + k2

= = KM

Michaelis-Menten ModelMichaelis-Menten Model• it is now possible to solve for the concentration of

the enzyme-substrate complex in this way

• or alternatively

[ES] =[E]T [S]KM + [S]

[E]T [S] - [ES][S]

[ES]= KM

= KM[ES]

[E]T [S] = [ES](KM + [S])

[E]T [S] - [ES][S]

Michaelis-Menten ModelMichaelis-Menten Model• in the initial stages, formation of product depends o

nly on the rate of breakdown of ES

• if substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T

• substituting k2[E]T = Vmax into the top equation gives

Vinit = k2[ES] = k2[E]T [S]KM + [S]

Vinit = Vmax = k2[E]T

Vmax [S]Vinit = KM + [S]

Michaelis-Menten ModelMichaelis-Menten Model• when [S]= KM, the equation reduces to

Vmax [S]V =

KM + [S]=

Vmax [S]

[S] + [S]=

(Presentation)

Michaelis-Menten ModelMichaelis-Menten Model• it is difficult to determine Vmax experimentally

• the equation for a hyperbola

• can be transformed into the equation for a straight line by taking the reciprocal of each side

Vmax [S]V =

KM + [S](an equation for a hyperbole)

KM + [S]

Vmax [S]=

KM [S]Vmax [S] Vmax [S]

Vmax [S] Vmax

Lineweaver-Burk PlotLineweaver-Burk Plot• which has the form y = mx + b, and is the formula for

a straight line

• a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax

• such a plot is known as a Lineweaver-Burk double reLineweaver-Burk double reciprocal plotciprocal plot

+ 1Vmax [S]

y m x + b

KM •

Lineweaver-Burk PlotLineweaver-Burk Plot

x intercept =

y intercept =1Vmax

slope =KMVmax

Significance of KSignificance of KMM and V and Vmaxmax

• KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E

• Vmax is the turnover number; moles of S that react to form product per mole of E per unit time

Acetylcholinesterase

Carbonic anhydrase

Catalase

Chymotrypsin

Turnover numbr

[(mol S)•(mol E)-1•s-1]

(mol•liter-1)

1.4 x 104

1.0 x 106

1.0 x 107

1.9 x 102

9.5 x 10-5

1.2 x 10-2

2.5 x 10-2

6.6 x 10-4

Enzyme InhibitionEnzyme Inhibition• Reversible inhibitorReversible inhibitor: a substance that binds to

an enzyme to inhibit it, but can be removed• competitive inhibitorcompetitive inhibitor: binds to the active (catalytic)

site and blocks access to it by substrate• noncompetitive inhibitornoncompetitive inhibitor: binds to a site other than

the active site; inhibits by changing the conformation of the enzyme

• Irreversible inhibitorIrreversible inhibitor: inhibition cannot be reversed• usually involves formation or breaking of covalent

bonds to or on the enzyme

Competitive InhibitionCompetitive Inhibition• substrate must compete with inhibitor for the active

site; more substrate is required to reach a given reaction velocity

• we can write a dissociation constant, KI for EI

E + S ES P+IEI

E+IEI KI =[E][I][EI]

Competitive InhibitionCompetitive Inhibition

• in a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the slope (and the x intercept) changes but the y intercept does not change

Vmax Vmax

No inhibition

In the presence of a competitive inhibitor

Vmax Vmax+ 11 +[I]

+ bm x

Competitive InhibitionCompetitive Inhibition

x interceptsy intercept =1Vmax

slope =KMVmax

No inhibition

Competitiveinhibition

KM 1 +[I]

Vmax1 +

KIslope =

Noncompetitive InhibitionNoncompetitive Inhibition• because the inhibitor does not interfere with binding

of substrate to the active site, KM is unchanged

• increasing substrate concentration cannot overcome noncompetitive inhibition

y = m x

In the presence of a noncompetitive inhibitor

Vmax Vmax+ 11 +

1 +[I]

Vmax Vmax

+ 1No inhibition

Noncompetitive InhibitionNoncompetitive Inhibition

x intercept y intercept =1Vmax

slope = KMVmax

No inhibition

Noncompetitiveinhibition

Vmax1 +[I]

slope =

y intercept =1Vmax

1 +[I]

Allosteric EnzymesAllosteric Enzymes• AllostericAllosteric: Greek allo = other + steric = shape• Allosteric enzymeAllosteric enzyme: an oligomer whose biologic

al activity is affected by other substances binding to it• these substances change the enzyme activity by al

tering the conformation(s) of its 4° structure

• Allosteric effectorAllosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an• allosteric inhibitor• allosteric activator

• Aspartate transcarbamylase (ATCase)Aspartate transcarbamylase (ATCase)

H2N-C-OPO32-

CH-CO2-H3N+

H2N-C-NH-CH-CO2-

H3PO42-

ATCaseCarbamoylphosphate

Aspartate

N-Carbamoylaspartate

-O-P-O-P-O-P-O-CH2O

O Series ofsteps

Cytidine triphosphate (CTP)

CTP inhibitsATCase!

-O-P-O-P-O-P-O-CH2O

Cytidine triphosphate (CTP)

-O-P-O-P-O-P-O-CH2O

Adenosine triphosphate (ATP)

an allosteric inhibitor of ATCase

an allosteric activator of ATCase

ATCase - ATCase - an allosteric enzymean allosteric enzyme

+ ATP (an allosteric activator)

+ CTP (an allosteric inhibitor

Control - no ATP or CTP

The Concerted ModelThe Concerted Model• Wyman, Monod, and Changeux - 1965• The enzyme has two conformations

• R (relaxed)R (relaxed): binds substrate tightly; the form active• T (tight)T (tight): binds substrate less tightly; the inactive for

m• in the absence of substrate, most enzyme molecules

are in the T (inactive) form• the presence of substrate shifts the equilibrium from

the T (inactive) form to the R (active) form • in changing from T to R and vice versa, all subunits

change conformation simultaneously; all changes are concerted

Concerted ModelConcerted Model• the binding of substrate to one enzyme subunit facili

tates binding of a second substrate to a second enzyme subunit

• allosteric inhibitors bind to and stabilize the T (inactive) form

• allosteric activators bind to and stabilize the R (active) form

Sequential ModelSequential Model• Koshland, Nemethy, and Filmer - 1966

• the binding of substrate induces a conformational change from the T form to the R form

• the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding

• a change of one subunit from T to R makes the same change easier in other subunits

• allosteric activation and inhibition also occur by the induced-fit mechanism

ZymogensZymogens• ZymogenZymogen: an inactive precursor of an enzyme;

cleavage of one or more covalent bonds transforms it into the active enzyme

• Chymotrypsinogen• synthesized and stored in the pancreas• a single polypeptide chain of 245 amino acid residue

s cross linked by five disulfide (-S-S-) bonds• when it is secreted into the small intestine, the diges

tive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin

ZymogensZymogens• the 15-unit polypeptide remains bound to -chymotr

ypsin by a single disulfide bond• -chymotrypsin catalyzes the hydrolysis of three of i

ts own peptide bonds to give -chymotrypsin• -chymotrypsin consists of three polypeptide chains

joined by two of the five original disulfide bonds• changes in 1?structure that accompany the change f

rom chymotrypsinogen to -chymotrypsin result in changes in 2?and 3?structure as well.

• -chymotrypsin is enzymatically active because of its 2?and 3?structure, just as chymotrypsinogen was inactive because of its 2?and 3?structure

The Active SiteThe Active Site1. Which amino acid residues on the enzyme are i

n the active site and catalyze the reaction?

2. What is the spatial relationship of the essential amino acids residues in the active site?

3. What is the mechanism by which the essential amino acid residues catalyze the reaction?

• As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Lys or Arg

ChymotrypsinChymotrypsin• Reaction with a model substrate

O2N OCCH3

O2N O-

CH3CO-O

p-Nitrophenylacetate

p-Nitrophenolate

Step 1 E +

E-OCH3

Step 2 E-OCH3

O+ H2O E +

Enzyme

An acyl-enzymeintermediate

ChymotrypsinChymotrypsin• DIPF inactivates chymotrypsin by reacting with

serine-195, which must be at the active site

Enz-CH2OH F-P-OCH(CH3)2

OCH(CH3)2

Diisopropylphospho-fluoridate

(DIPF)

Serine-195

Enz-CH2 O-P-OCH(CH3)2

OCH(CH3)2A labeled enzyme

(inactive)

ChymotrypsinChymotrypsin• TPCK labels Histidine-57

Enz-CH2

C6H5CH2-CH-C-CH2Cl

N-Tosylamido-L-phenylethylchloromethyl ketone (TPCK)

(Tsyl = tosyl group)

Histidine-57

Enz-CH2

C6H5CH2-CH-C-CH2

ChymotrypsinChymotrypsin• because Ser-195 and His-57 are required for activity,

they must be close to each other in the active site• the results of x-ray crystallographic show the definit

e arrangement of amino acids at the active site• in addition to His-57 and Ser-195, Asp-102 is also inv

olved in catalysis at the active site

• The mechanism by which chymotrypsin catalyzes the hydrolysis of amide bonds is shown in Figure 5.19

Catalytic MechanismsCatalytic Mechanisms

Cys-SH Cys-S-

Lys-NH3+ Lys-NH2

Glu-CO2H Glu-CO2-

Ser-CH2OH Ser-CH2O-

His-CH2N

Tyr OH Tyr O-

Proton Donor Proton AcceptorType of Group

sulfhydrylaminocarboxylhydroxyl

imidazole

phenol

Catalytic MechanismsCatalytic Mechanisms• Lewis acid/base reactions

• Lewis acidLewis acid: an electron pair acceptor• Lewis baseLewis base: an electron pair donor

• Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes• carboxypeptidase A requires Zn2+ for activity

Catalytic MechanismsCatalytic Mechanisms• Zn2+ of carboxypeptidase is complexed with

• the imidazole side chains of His-69 and His-196 and the carboxylate side chain of Glu-72

• it activates the carbonyl group for nucleophilic acyl substitution

NCH-CO2-H

Zn(II)

Lewis acid

Lewis base

Zn(II) of carboxypeptidase iZn(II) of carboxypeptidase is complexed withs complexed with

• the imidazole side chains of His-69 and His-196 and the carboxylate side chain of Glu-72

• it activates the carbonyl group for nucleophilic acyl substitution

• (Please see p186)

CoenzymesCoenzymes• CoenzymeCoenzyme: a nonprotein molecule or ion that t

akes part in an enzymatic reaction and is regenerated for further reaction• metal ions• organic compounds, many of which are vitamins or

are metabolically related to vitamins

Coenzyme Reaction TypeVitamin Precursor

Biotin

Coenzyme A

Flavin coenzymes

Lipoic acid

Nicotinamide coenzymes

Pyridoxal phosphate

Tetrahydrofolic acid

Thiamine pyrophosphate

Carboxylation

Acyl transfer

Oxidation- reduction

Acyl transfer

Oxidation- reduction

Transamination

One-carbon transfer

Aldehyde transfer

Pantothenic acid

Riboflavin (B2)

Niacin

Pyridoxine (B6)

Folic acid

Thiamine (B1)

Metal Ion Enzyme

Fe2+ or Fe3+

Peroxidase

Cytochrome oxidase

DNA polymerase

Hexokinase

Arginase

Pyruvate kinase

Urease

Nitrate reductase

Glutathione peroxidase

NADNAD++/NADH/NADH• Nicotinamide adenine dinucleotide (NAD+) is a

biological oxiding agent

a -N-glycoside bond

The plus sign on NAD+represents the positive charge on this nitrogen Nicotinamide,

derivedfrom niacin;

-O-P-O-CH2

NADNAD++/NADH/NADH• NAD+ is a two-electron oxidizing agent, and is

reduced to NADH

++ H+ + 2e-

(oxidized form)NADH

(reduced form)

NADNAD++/NADH/NADH• NAD+ is involved in a variety of enzyme-

catalyzed oxidation/reduction reactions, two of which are

O+ 2H+ 2e-

A secondary alcohol

A ketone

+ H2O C OH

2H+ 2e-

An aldehyde A carboxylic acid

NADNAD++/NADH/NADH

reduction

oxidation

An electron pair is added to nitrogen

Pyridoxal PhosphatePyridoxal Phosphate

CH2OHHO

Pyridoxal

CH2OPO-HO

CH2NH2

Pyridoxamine phosphate

CH2OHHO

CH2NH2

Pyridoxamine

CH2OPO-HO

Pyridoxal phosphate

Pyridoxal PhosphatePyridoxal Phosphate• Pyridoxal and pyridoxamine phosphates are inv

olved in the transfer of amino groups

-O2CCH2CH2CHCO2-

GlutamateCH3CCO2

Pyruvate+

-O2CCH2CH2CCO2-

-Ketoglutarate

CH3CHCO2-

Alanine

transaminase,pyridoxal phosphate

Lecture6enzy advance

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