Review Enzyme “constants” –K m –V max –k cat –k cat /K m –K i Reversible inhibition –Impact on K m and V max for each Irreversible inhibition –I combines/binds.

Review

• Enzyme “constants”– Km

– Vmax

– kcat

– kcat/Km

– Ki

• Reversible inhibition– Impact on Km and Vmax for each

• Irreversible inhibition– I combines/binds to E to form a very stable

complex

E + S + I ES + I E + P

EI ESI

Question….

• Methanol (wood alcohol) is highly toxic because it is converted to formaldehyde in a reaction catalyzed by the enzyme alcohol dehydrogenase:

• NAD+ + methanol NADH + H+ + formaldehyde

• Based on enzyme inhibition, what’s a possible treatment for methanol poisoning?

Irreversible inhibition

• Suicide/mechanism based inhibitor– A few chemical steps are carried out– Compound converted to reactive intermediate that

irreversibly reacts with enzyme– Used in drug design

• Potential for high potency• Typically very specific for the enzyme: few side effects

Chymotrypsin: Specific enzyme mechanism

• Protein structure determines function

Chymotrypsin

• Protease specific for bonds adjacent to aromatic AA• Hydrolysis reaction

– But: enzyme doesn’t catalyze direct attack by water

• Stabilization of E-TS• General acid/base and covalent catalysis• Two phases to the reaction:

1.AcylationCleavage of peptide bond and formation of ester with enzyme

2.DeacylationHydrolysis of ester and enzyme regenerated

Kinetics → Mechanism

Fast phase (burst phase/pre-steady state)

Kinetics → Mechanism

Histidine must be deprotonatedfor rxn to occur

Ile (N-term) must beprotonated for substrateto bind

• Catalytic triad components– Nucleophile-Ser195

– His57 (general base)– Asp102 (stabilizes +

charge on His)

• Oxyanion hole– Stabilizes O- in

tetrahedral intermediate

Chymotrypsin: “Catalytic triad” protease

Chymotrypsin

Chymotrypsin mechanism

Chymotrypsin mechanism

Chymotrypsin

• Formation of acyl-enzyme intermediate– Covalent bond between enzyme and

substrate/transition state• Actual breaking of the peptide bond

• Deacylation– Activation of water to break the enzyme-

substrate bond• Release of the rest of the substrate protein

Enzymes and regulation

• Activity can modulated by several factors– Maximize biological efficiency: stop or speed-up a

pathway under appropriate conditions

1. Allostery

2. Reversible covalent modification– Addition of sugars, phosphates, adenine, acetate, etc.

3. Reversible binding of other, regulatory, proteins

4. Proteolytic cleavage

Allostery

• Modulation of equilibrium between more/less active forms

Allostery• Aspartate transcarbamoylase

– Pyrimidine synthesis• Binding of modulator to regulatory

subunit inhibits activity• CTP negative modulator• Feedback (product inhibition)

Allostery: a case where M-M doesn’t quite work

Sigmoidal V vs S curves

Change in Vmax, not K0.5Change in K0.5, not Vmax

Covalent modification

• Phosphorylation, adenylation, uridylation, methylation…..

– Change electrostatic interactions/repulsion• Phosphorylation of serines adds a negative charge• Acetylation of lysines removes a positive charge

– Conformational change → turns ‘on’ or ‘off’

Reversible phosphorylation

• Phosphate added by kinases

• Phosphate removed by phosphatases

• Typically serine/threonine or tyrosine, sometimes histidine

Phosphorylation of glycogen phosphorylase

• Glycogen phosphorylase ‘a’ and ‘b’– Converts glycogen

to glucose 1-phosphate for energy

Proteolytic cleavage

• Synthesis as ‘zymogen’ (inactive enzyme precursor)– Chymotrypsinogeninactive →

chymotrypsinactive

– Cleavage → conformational change that exposes active site

– Mechanism used for other proteins as well

• Procollagen collagen

• Fibrinogen fibrin

• Proinsulin insulin