6p1-1 chapter 6: outline-1 properties of enzymes classification of enzymes enzyme kinetics...

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Chapter 6: Outline-1Properties of EnzymesClassification of EnzymesEnzyme Kinetics

Michaelis-Menten KineticsLineweaver-Burke PlotsEnzyme Inhibition

CatalysisCatalytic MechanismsCofactors

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Chapter 6: Outline-2Catalysis cont.

Temperature and pHDetailed Mechanisms

Genetic ControlEnzyme RegulationCovalent ModificationAllosteric RegulationCompartmentation

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IntroductionThe proteins which serve as enzymes,

Mother Nature’s catalysts, are globular in nature. Because of their complex molecular structures, they often have exquisite specificity for their substrate molecule and can speed up a reaction by a factor of millions relative to an uncatalyzed reaction. This presentation will describe how enzymes function.

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6.1 Properties of EnzymesA catalyst enhances the rate of reaction

but is not permanently altered.

Catalysts work by decreasing the activation energy for a reaction.

The structure of the active site of the enzyme (shape and charge distribution) is used to optimally orient the substrate for reaction.

The energy of the enzyme-substrate complex is then closer to the TS.

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Activation Energy, Eact

An enzyme speeds a reaction by lowering the activation energy. It does this by changing the reaction pathway.

Reaction progress

Free Energy

Products

Reactants Free energy change (G) for the reaction

Transition state

Activationenergy, G

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Activation Energy-2An enzyme lowers the activation energy

but it does not change the standard free energy change (G) for the reaction nor the Keq.

A catalyst cannot make an endergonic reaction exergonic or vice versa.

Most enzymes are temperature/pH sensitive and will not work outside their normal temperature/pH range because the enzyme is denatured.

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Enzymes ModelsIn the lock-and-key model, the enzyme is

assumed to be the lock and the substrate the key. The two are made to fit exactly. This model fails to take into account the fact that proteins can and do change their conformations to accommodate a substrate molecule.

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Enzymes Models-2The induced-fit model of enzyme action

assumes that the enzyme conformation changes to accommodate the substrate molecule. Eg.

Conformationchanges

Enzyme andsubstrate donot “fit”.

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6.2 Classification of EnzymesThe International Union of Biochemistry

(IUB) classifies and names enzymes according to the type of chemical reaction it catalyzes.

Enzymes are assigned a four-number class and a systematic two-part name.

A shorter recommended name is also suggested.

Alcohol dehydrogenase is:alcohol:NAD+ oxidoreductase(E.C. 1.1.1.1)

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Enzyme Classes1. Oxidoreductases catalyze redox

reactions. Eg. Reductases or peroxidases

2. Transferases transfer a group from one molecule to another. Eg. Transaminases, transcarboxylases

3. Hydrolases cleave bonds by adding water. Eg. Phosphatases or peptidases

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Enzyme Classes-24. Lyases catalyze removal of groups to

form double bonds or the reverse. Eg. decarboxylasaes or synthases

5. Isomerases catalyze intramolecular rearrangements. Eg. epimerases or mutases

6. Ligases bond two molecules together. Many are called synthetases. Eg. carboxylases

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Enzyme Classes-3: Examples

Class Example Reaction

1 alc dehy-

drogenase

2 hexokinase glucose + ATP glucose-6-phosphate

+ADP

3 chymotrypsin polypeptide + H2O

peptides

CH3CH2OH CH3CH

O

+ NAD+ + NADH + H+

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Enzyme Classes-3: ExamplesClass Example Reaction

4 pyruvate

decarboxylase

5 alanine

racemase

D-alanine L-alanine

6 pyruvate

carboxylase

CH3CO

CO

O CH3CHO

+ H+ + CO2

CH3CO

CO

O

O CO

CH2CO

CO

O

+ HCO3-

ATP ADP+Pi

_

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6.3 Enzyme KineticsKinetics is the field of chemistry that

studies the rate and mechanism of a reaction.

Rates are usually measured in terms of how many moles of reactant or product are changed per time period.

A mechanism is a detailed step-by-step description of how a reaction occurs at the molecular level.

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The Rate Equation-1A P

Init. Rate = vo = - A] or [P]

t t

= change in, [A] = molarity and t is time.

Disappearance of reactants is negative so the quantity has a negative sign to make all rates positive.

First order: Rate = A] = k[A]1

t

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The Rate Equation-2

Rate = k [A]x The rate equals the experimentally determined rate constant, k, times the concentrations of A to some experimentally determined power, x. Values for x are frequently 0, 1 or 2.

ALL RATE EQUATIONS ARE DETERMINED EXPERIMENTALLY!!

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The Rate Equation-3A + B P

Init. Rate = vo = - A] or -B] or [P]

t t t

Rate = -k[A]1 [B]1 (overall second order)

If B is water (in large excess) then the reaction appears to be first order in A and is said to be pseudo first order.

Rate’ = k’[A]1 [B]0 = k’[A]1

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The Rate Equation-4A + A P

Second order: Rate = A] = k[A]2

t

Two molecules of A collide to give P.

A PZero order: Rate = A] = k[A]0

t Concentration of A has no effect on rate.

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Rate Equation Example

For the reaction Gly-Gly + H2O 2 Gly

Use the data to determine x and y in therate equation: Rate = k[di-G]x[H2O]y

[di-Gly] [H2O] Rate, Ms-1 x 10-2

a) 0.1 0.1 1

b) 0.2 0.1 2

c) 0.1 0.2 2Water constant and di-Gly doubled (a+b). Rate doubles. X=1Di-Gly constant and water doubles (a+c). Rate doubles. Y=1

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Enzyme Rxns: Type 1Chymotrypsin cleaves proteins at the

COOH end of aromatic side chain AAs.

At low substrate concentrations, the reaction is first order in substrate.

As the concentration of substrate increases, the order changes and approaches zero.

A graph of velocity vs substrate conc. is hyperbolic. (See graph, #22)

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Enzyme Rxns: Type 1 Chymo-

trypsin: Hyperbolic plot

First order

Zero order

Conc. At ½ max velocity

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Enzyme Rxns: Type 2Aspartate transcarbamoylase (ATCase)

catalyzes the reacton between aspartate and carbamoyl phosphate.

This reaction leads ultimately to the synthesis of nucleobases needed for DNA and RNA synthesis.

Velocity as a function of aspartate concentration gives a sigmoidal plot. (See #24)

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Enzyme Rxns: Type 2

ATCase:Sigmoidal plot

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Enzyme Rxns-cont.The plots for cymotrypsin and aspartate

transcarbamoylase should remind us of the oxygen binding curves for myoglobin and hemoglobin respectively.

Thusly, chymotrypsin is a nonallosteric and ATCase is an allosteric enzyme.

We need two different models to explain these enzymes behaviors.

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Michaelis-Menten Kinetics-1

M-M kinetics explains the behavior of nonallosteric enzymes. It assumes an enzyme-substrate complex is formed.

At low substrate concentrations, the reaction is first order with respect to substrate.

At high substrate concentrations, the enzyme is saturated with substrate. The order is

zero and a Vmax occurs.

E + S E-S E + Pk1

k2

k3

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Michaelis-Menten Kinetics-2At low P concentrations (initial rate)

Assumed: k2 negligible vs k1

Rate of formation of ES equals rate of degradation.

Rate = P/t = k3[ES]

k1[E][S] = (k2 + k3)[ES] (Steady State)

[ES] = [E][S] (k2 + k3)/k1

Km = (k2 + k3)/k1

Michaelis constant

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Michaelis-Menten Kinetics-3Michaelis and Menten also derived what

is now known as the Michaelis-Menten equation.

An enzymes’s kinetic properties can be used to determine its catalytic efficiency. (Next slide.)

v = Vmax[S][S] + Km

Vmax = max velocity

The lower the Km, the greater the affinity for complex formation.

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Michaelis-Menten Kinetics-4kcat = Vmax/[Et] = turnover number

kcat = molecules of S converted to product per unit time with enzyme saturated

[Et] = total enzyme concentrationV= (kcat / Km) [E][S]

(kcat / Km) is a rate constant where

[S]<< Km and the constant reflects thecombined effect of binding andcatalysis.

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Michaelis-Menten Kinetics-5

Turnover numbers for some enzymes follow. They vary greatly!!

Enzyme kcat (s-1)

Catalase 10,000,000

Chymotrypsin 190

Lysozyme 0.5

Note: catalyse turns over 10 milliion molecules of substrate per sec!!

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Michaelis-Menten Kinetics-6Substrate concentration at ½ Vmax is

termed the KM (Michaelis constant) for

the reaction.

KM is difficult

to measure bythis method as

Vmax must be

estimated.A linear plotgives betterresults.

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Lineweaver-Burk PlotA Lineweaver-Burk plot for nonallosteric

enzymes gives a straight line and better data to determine KM.

=KM

Vmax

1Vmax

+ 1[S]

1V

In the form y = mx + b:1/V is y (V is the measured velocity (rate) of

the reaction), 1/[S] is x, KM/Vmax is the slope

and 1/Vmax is the y intercept.

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Lineweaver-Burk Plot

A L-B plot of 1/V vs 1/[S] is shown below

1V

1/[S]0

y intercept is1Vmax

x intercept is-1KM

slope isKM

Vmax

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Enzyme InhibitionInhibitors interfere with enzyme action. They may be reversible or irreversible.The three kinds of reversible inhibitors

are competitive uncompetitive and noncompetitive .

A competitive inhibitor looks structurally like the substrate and binds to the enzyme at the active site.

An uncompetative inhibitor binds only to the enzyme-substrate complex.

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Enzyme Inhibition-2A noncompetitive inhibitor does not look

like substrate and binds at a site other than the active site.

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Competitive Inhibitor

E + S E-S E + P+

EI + S

I

NR

K1

K2

K3

K12 K11

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Competitive Inhibitor-2Since a competitive inhibitor competes

with substrate for the active site, its influence can be negated with large concentrations of substrate. Thus the Vmax remains constant.

Since the velocity is slower compared to normal substrate concentrations, the slope of the L-B line increases and the KM increases.

The effect of a competitive inhibitor on a L-B plot is shown on slide 42.

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Uncompetitive Inhibitor

E + S E-S E + P+

EIS

I

K1

K2

K3

K12 K11

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Uncompetitive Inhibitor-2Since an uncompetitive inhibitor binds

only to the enzyme-substrate complex, adding more substrate will increase the rate but not to the original values without inhibitor.

Commonly observer when the enzyme binds to more than one substrate.

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Noncompetitive Inhibitor

E + S E-S E + P+

ES

I

K1

K2

K3

I+

EI + S NR

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Noncompetitive Inhibitor-2For a noncompetitive inhibitor, the

velocity of the reaction is slowed at all substrate concentrations. Thus the Vmax is permanently lowered.

The slope of the L-B line increases but KM stays constant.

The effect of a noncompetitive inhibitor on a L-B plot is shown on slide 42.

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Kinetics: InhibitionCompetitive:Vmax sameKM changes

Noncompetitive:Vmax changesKM same

1V

1/[S]0

No inhibitor

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The kinetic behavior of allosteric enzymes, catalysis in general, and specific enzyme mechanisms are

discussed in the next slide series:

Enzymes Part 2