2. Review Enzyme “constants” Reversible inhibitionKm
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

3. 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?

4. 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

5. Chymotrypsin: Specific enzyme mechanism
Protein structure determines function

6. 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:
Acylation
Cleavage of peptide bond and formation of ester with enzyme
Deacylation
Hydrolysis of ester and enzyme regenerated

7. Kinetics → Mechanism
Fast phase (burst phase/pre-steady state)

8. Kinetics → Mechanism Histidine must be deprotonated for rxn to occur
Ile (N-term) must be
protonated for substrate
to bind

9. Chymotrypsin: “Catalytic triad” protease
Catalytic triad components
Nucleophile-Ser195
His57 (general base)
Asp102 (stabilizes + charge on His)
Oxyanion hole
Stabilizes O- in tetrahedral intermediate

10. Chymotrypsin

11. Chymotrypsin mechanism

12. Chymotrypsin mechanism

13. Chymotrypsin Formation of acyl-enzyme intermediate Deacylation
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

14. Enzymes and regulation
Activity can modulated by several factors
Maximize biological efficiency: stop or speed-up a pathway under appropriate conditions
Allostery
Reversible covalent modification
Addition of sugars, phosphates, adenine, acetate, etc.
Reversible binding of other, regulatory, proteins
Proteolytic cleavage

15. Allostery
Modulation of equilibrium between more/less active forms

16. Allostery Aspartate transcarbamoylase Pyrimidine synthesis
Binding of modulator to regulatory subunit inhibits activity
CTP negative modulator
Feedback (product inhibition)

17. Allostery: a case where M-M doesn’t quite work
Sigmoidal V vs S curves
Change in K0.5, not Vmax
Change in Vmax, not K0.5

18. 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’

19. Reversible phosphorylation
Phosphate added by kinases
Phosphate removed by phosphatases
Typically serine/threonine or tyrosine, sometimes histidine

20. Phosphorylation of glycogen phosphorylase
Glycogen phosphorylase ‘a’ and ‘b’
Converts glycogen to glucose 1-phosphate for energy

21. 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