Biochemistry

Lecture 8

Why Enzymes?• Higher reaction rates• Greater reaction specificity• Milder reaction conditions• Capacity for regulation

COO

OH

O COO

COO

O COO

NH2

OOCCOO

O

OH

OH

COO

NH2

COO

-

-

-

-

-

-

--

Chorismate mutase

• Metabolites have many potential pathways of decomposition

• Enzymes make the desired one most favorable

Enzymatic Substrate Selectivity

No binding

OOC NH3

H

OOC NH3

H

HNH

HOH

OH

H

OH

CH3

OOC NH3

H

OH

--

-

+

+

+

Binding but no reaction

Example: Phenylalanine hydroxylase

How to Lower G?Enzymes organizes reactive groups into

proximity

How to Lower G?Enzymes bind transition states best

How is TS Stabilization Achieved?

– acid-base catalysis: give and take protons

– covalent catalysis: change reaction paths

– metal ion catalysis: use redox cofactors, pKa shifters

– electrostatic catalysis: preferential interactions with TS

End result? Rate enhancements of 105 to 1017!

How is TS Stabilization Achieved?

– covalent catalysis: change reaction paths

CH3O

O

CH3

O

CH3O

O

CH3O

O

H+- -+

H2O

slow + 2

CH3O

O

CH3

O

N

CH3O

O

N CH3

O

OH H

N CH3

O

OHN

CH3O

O

H+

..fast

-+ +

..

+

-

-+..

Enzyme Kinetics

• Kinetics is the study of the rate at which compounds react

• Rate of enzymatic reaction is affected by– Enzyme– Substrate– Effectors– Temperature

How to Do Kinetic Measurements

What equation models this behavior?

Michaelis-Menten Equation

Meaning of Vmax and Km

Simple Enzyme Kinetics

• The final form in case of a single substrate is

• kcat (turnover number): how many substrate molecules can one

enzyme molecule convert per second

• Km (Michaelis constant): an approximate measure of

substrate’s affinity for enzyme

• Microscopic meaning of Km and kcat depends on the details of

the mechanism

][

]][[

SK

SEkv

m

totcat

Two-substrate Reactions

• Kinetic mechanism: the order of binding of substrates and release of products

• When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms

– Sequential mechanism– Ping-Pong (Double Displacement) mechanism

Distinguishing Mechanism

Ping-PongTernary Complex

Enzyme Inhibition

Inhibitors are compounds that decrease enzyme’s activity

• Irreversible inhibitors (inactivators) react with the enzyme- one inhibitor molecule can permanently shut off one enzyme molecule- they are often powerful toxins but also may be used as drugs

• Reversible inhibitors bind to, and can dissociate from the enzyme - they are often structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme

• Reversible inhibitor can bind:

– To the free enzyme and prevent the binding of the substrate

– To the enzyme-substrate complex and prevent the reaction