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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
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