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Review Enzyme “constants” Reversible inhibition

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2 Review Enzyme “constants” Reversible inhibition
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

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

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