BT-202Netaji Subhas Institute of Technology,

Dwarka, New Delhi.

Dr. Amita Pandey

Sept 5, 2011

• Enzyme kinetics

• Catalytic mechanisms

• Regulation of enzyme activity

• Enzyme inhibition

• Louis Pasteur (1850s): ferments, vitalism

• Eduard Buchner (1897): fermentation is done by molecules

• Frederick W. Kuhne: coined the name enzyme

• James Sumner (1926): isolated urease and said enzymes are made up of proteins.

• J.B.S. Haldane: weak interactions between enzyme and substrate are important for catalysis

Enzymes

Definition;Definition;

•Chemical reactions in cells requirespecific catalysis.

•Enzymes are proteins which performthis function.-12kDa - 1,000kDa or more -larger than their substrate

Enzymes Enzymes

• Metabolite acted upon is called the enzyme’s substrate.

• Active site

-some residues involved in binding substrate

-others catalyze reaction

Cofactors

for some reactions, the amino acids are not powerful enough for catalysis. Enzymes overcome this by incorporate additional factors.

-metal ions as cofactors Zn2+, Fe2+, Cu2+

-coenzymes are organic cofactors

Prosthetic Group

Coenzyme or metal ion cofactor bound to enzyme either tightly or covalently.

-holoenzyme

-apoenzyme

Cofactor and Coenzymes

Properties of Enzymes

• Enzymes are excellent catalysts-speeding up reactions 108 to 1020 fold

• Not used up during a reactions • Specificity for substrate

-absolute (eg., DNA polymerase)

-broad range (eg., synthesis of secondary metabolites)

• Regulated- some enzymes can sensemetabolic signals.

Classification of EnzymesClassification of Enzymes

• named and classified according to the substrate acted upon and the reaction catalyzed.

• trivial names- end in –ase. (eg., urease, hexokinase.

• based on a formal systemic catalog (IUB) with six major classifications.

Reaction Rates and theReaction Rates and theTransition StateTransition State

• In order to react, the molecules involvedare distorted, strained or forced to havean unlikely electronic arrangement.

• That is the molecules must pass through ahigh energy state.

• This high energy state is called the transition statetransition state.

• The energy required to achieve it is calledthe activation energyactivation energy for the reaction.

The higher the freeenergy change for the transition barrier, the slower the reaction rate.

Enzymes lower energybarrier by forcing thereacting moleculesthrough a differenttransition state.

Eg.,

C12H22O11 + 12O2 12CO2 + 11H2O

Reaction IntermediatesTransient chemical species formed during a rxn

Rate limiting step When many steps are involved the overall rate of the Reaction is determined by the step which has the Highest activation-energy

Relationship between reaction equilibrium and

free energy Equilibrium constant is directlyrelated to free energy

Large negative free energy favors reaction

Rate of a reaction

Unimolecular or first order reaction;

V=k[S]Units are s-1

Rate for second order reaction;

V= k[S1][S2]

for transition statek = kT / h e-ΔG/RT

Lower activation energy means faster rxn rates

How enzymes do it?

Binding energy (ΔGB)Energy derived from enzyme substrate interaction.-transient covalent bond in the active site-non-covalent interactions to form ES

Role of binding energy in catalysis

k = kT / h e-ΔG/RT

ΔG+ must be lowered by 5.7kJ/mol to accelerate first order reaction.

•Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate.

• Entropy reduction

• Desolvation of substrate

• ES complex

• Enzyme undergoes conformation

changes i.e. induced fit, postulated

by Daniel Koshland (1958)

Modes of Enzymatic Modes of Enzymatic Enhancement of RatesEnhancement of Rates

• Involve transient covalent interactions with a substrate or group transfer to or from a substrate

• General acid-base catalysis-good proton donors & acceptors positioned just right.

• covalent catalysis-unstable intermediate

• metal ion catalysis-electron donor or acceptor

•At low concentrations of [S] Vo increases almost linearly

•At higher [S] concentration Vo increases by smaller amounts in response to increase in [S]

•Finally increase in Vo is negligible as [S] increases

Enzyme kinetics

Steady state kineticsSteady state kinetics

• rate stops increasing or plateaus because the complex ES becomes filled at high [S]

• Pre-steady state and steady state

Introduced by Briggs and Haldane

Michaelis-Menten ModelMichaelis-Menten Model

• Rate equation

Lineweaver-Burk equation

Double reciprocal plot

Interpreting Vmax and Km

Steady state kinetics is the means by which biochemists can compare and characterize The catalytic efficiencies of enzymes.

Kcat it is the limiting rate of any enzyme catalyzed reaction. Eg.,

Kcat is first order rate constant has units s-1. It is also called Turnover number (the number of substrate molecules converted to product).

Specificity constant compares the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme.

Vo = kcat / Km [Et][S]

-V0 depends upon [Et] and [S]-kcat/Km is a second order rate constant-units M-1s-1

Reactions with more than one substrate

Learning check!

1. In a simple enzyme catalyzed rxn., the sum of which two chemical species is strictly constant?

[E] + [ES][E] + [P][S] + [P][ES] + [P]

2. For a simple enzymatic rxn., what is the value of The initial velocity when S=5km?

5 Vmax5/2 Vmax4/5 Vmax5/6 Vmax

Match the followinga)Michaelis complex The velocity at t=0b)Michaelis constant Km Also known as turnover numberc)Initial velocity The enzyme substrate complexd)Maximal velocity The velocity when the enzyme is saturated with substratee)Catalytic velocity The conc. of substrate @ which the velocity is half maximal

rxns. are enzyme reactions with two substrates. Bisubstrate

In Lineweaver-Burk plot of a simple enzymatic rxn. , what is the value of the y-intercept @ 1/Vo axis?

Vmax1/VmaxKm1/Km

Regulatory enzymes

Exhibit increased or decreased catalytic activityin response to certain signals.

-Reversible non-covalent modulation-allosteric modulators

-small metabolites or cofactors

-Reversible covalent modification

-inhibition with separate regulatory protein

-proteolytic cleavage

Allosteric enzymes

-Homotropic-Heterotropic

Heterotropic Allosteric Modulation

In multienzyme pathways the regulatory enzyme is inhibited by the end product.

Eg., L-Threonine to L-Isoleucine in bacteria.

Kinetics of Allosteric enzymes

-sigmoid curve is observed

-substrate concentration is represented by [S]0.5 or K0.5

homotropic allosteric enzymes

-reflects cooperative interaction between protein molecules

-small changes in conc. of modulator can be associated with large changes in activity

Heterotropic Allosteric enzymes

-Activation may cause increased velocity for fixed K0.5

-Negative modulator cause decreased velocityfor fixed K0.5

-increased Vmax with little change in substrate

Learning check!

Match the following

Competitive Inhibiton decreases Km and VmaxUncompetitive inhibition decreases [E]TMixed inhibition increases KmInactivation decreases Vmax, while Km

may increase or decrease

Mixed inhibition is characterized by two dissociation constants for the inhibition.TrueFalse

Reversible covalent modification

How do the modulators act?

phosphatasekinase

-oxygen atom of phosphoryl group undergo H-bond formation

-repulsion of neighboring residues with negative charges

Glucose-6-

phosphate

Glucose-6-

phosphate

ATP synthesis in muscles

Free glucose in liver

-phosphorylation occurs in structural motif calledconsensus sequence

-AA sequence is not the only factor which determines phosphorylation

Regulation by proteolytic cleavage

-zymogen an inactive precursoris cleaved to form the activeenzyme

-precursors are also called proproteineg., collagen, fibrin, and thrombin

Enzyme inhibitors

Interfere with catalysis, slowing or halting enzymaticreactions.

-Reversible Inhibition-competitive

Since it is reversible so increasing the concentration of substrateremove inhibition.

Uncompetitive inhibition

Lower the measured V max

Apparent Km also decreases

Mixed Inhibition

Irreversible Inhibition

DIFP Diisopropylfluorophosphate

Suicide inactivators such compounds are inactive untill they bind active site of a specific enzyme.