Enzyme Features

• Increase rate of reaction• Active site binds substrate• Unchanged by overall reaction

Enzyme Classes

Reaction Equilibria

• G is the free energy difference• G depends upon [S] and [P]• Gº is the standard free energy change

Reaction: S ↔ P

G = Gº + RT ln ([P]/[S])

G Direction<0 S → P0 equilibrium>0 P → S

Energetics Of Catalysis

G

G‡)

G ‡)

• G determines direction of reaction

• G‡ determines rate of reaction• Enzymes lower G‡

but do not alter G

Reaction: S ↔ ST ↔ P

Energetics Of Coupled Reactions

ammonia + glutamic acid → glutamine (unfavorable)ATP → ADP + Pi (favorable)

• Unfavorable reaction coupled to favorable one

• Net reaction is favorable• For example, coupled reactions

catalyzed by glutamine synthase

Chemistry Of Catalysis

How enzymes accelerate reaction rates

• Orient substrate(s)• Stabilization of transition state• Acid-base catalysis• Covalent catalysis

Stabilization Of Transition State

• Enzyme binding lowers energy of reaction intermediates

Acid-base Catalysis

• Acidic residue tends to donate proton• Basic residue tends to take up proton• Pair with atoms in substrate and alter electron distribution

Covalent Catalysis

Activated serine forms covalent bond with substrate • Serine protease mechanism:

Temperature Vs. Reaction Rate

• Increase of temperature increases velocity

• Denatured at high temperature

pH Vs Reaction Rate

• Optimum pH reflects different groups being protonated or unprotonated

Substrate Conc Vs Reaction Rate

• Increase of substrate concentration increases reaction rate until Vmax

• At Vmax enzyme is saturated

Michaelis-menton Kinetics

(Vmax [S])

(Km + [S])v0 =

v0 = initial reaction velocityVmax = maximal velocityKm = Michaelis constant[S] = substrate concentration

Reaction: E + S ES E + P

• Km reflects affinity of E for S• v0 directly proportional to [E]

Assumptions Of Michaelis-Menton Equation

• [E] << [S]• Steady-state assumption: [ES] does not change with time• Initial velocity is measured

Reaction: E + S ES E + P

Order Of Reaction

• If [S]<<Km, v0

proportional to [S]• If [S]>>Km, v0 nearly

independent of [S]

Lineweaver-burke Plot1v0

Km

Vmax[S] Vmax

1= +

• double reciprocal plot

Competitive Inhibitor

• Binds to same site as substrate

• Inhibition counteracted by increasing [S]

Effect Of Competitive Inhibitor On KM & VMAX

• Km increased, Vmax unchanged

Noncompetitive Inhibitor

• Binds to different site than substrate

• Inhibition not counteracted by increasing [S]

Effect Of Noncompetitive Inhibitor On KM & VMAX

•Vmax decreased, Km unchanged

Regulation Of Enzyme Activity

• Allosteric effectors• Phosphorylation• Activation of zymogens

Allosteric Enzymes

• Allosteric effectors bind regulatory site• Conformational change• Positive and negative effectors

Positive Effectors

• Binding to regulatory site increases catalysis at active site

Negative Effectors

• Binding to regulatory site inhibits catalysis at active site

Feedback Inhibition

• End product often negative effector for enzyme of initial step

Cooperative Allosteric Effects

• Symmetrical assemblies of identical subunits

• Cooperative binding of effector• Sharper response of enzyme

activity

Regulation By Phosphorylation

• Reversible covalent attachment of phosphate to serine, threonine or tyrosine

• Can alter activity

Coenzymes

• Small organic molecules• Binding site unique from substrate• Provide essential chemical group• Chemically changed by reaction

ATP

• Transfer of high energy phosphate• Modulator of enzyme activity

Nicotinamide Adenine Dinucleotide

• Derived from nicotinic acid (niacin)

• Adenosine with pyrophosphate linkage to ribose and nicotinamide

• Oxidation-reduction reactions• Niacin deficiency leads to pellagra

Riboflavin Coenzymes

• FAD = adenosine linked to riboflavin• FMN = phosphate linked to riboflavin• Oxidation-reduction reactions

Thiamine Pyrophosphate• Derived from thiamine

(vitamin B1)• Transfer of activated

aldehyde unit• Transketolase, pyruvate

dehydrogenase, -ketoglutarate dehydrogenase

• Thiamine deficiency leads to Beriberi (alcoholics)

Tetrahydrofolate

• Derived from folic acid• One carbon transfers,

example dTMP synthesis

• Folic acid deficiency leads to macrocytic (megaloblastic) anemia

Coenzyme B12 (Cobalamine)

• Corrin ring with central cobalt• Cobalt coordinated in six positions

Coenzyme B12 Reactions

• B12 deficiency leads to pernicious anemia

• Methylmalonyl CoA mutase reaction

• Methionine synthase reaction; THF trap

Coenzyme SummaryCoenzyme Reaction type Vitamin Consequences

precursor of deficiency

ATP Phospho transfer

NAD+/NADP+ Oxidation-reduction Nicotinic acid Pellagra(niacin)

FAD/FMN Oxidation-reduction Riboflavin (B2)

TPP Aldehyde transfer Thiamine (B1) Beriberi

Tetrahydrofolate Transfer one-carbon Folic Acid Anemiaunits

Coenzyme B12 Transfer methyl groups, B12 Anemia isomerization

Isoenzymes

• Different enzymes that catalyze same reaction• Often have different tissue distributions

Isoenzyme Analysis

• Creatine kinase- three isoenzymes from associations of two subunits• Distinguished based on charge by non-denaturing electrophoresis• Diagnosis of myocardial infarction