Transcript very stable
Review • Enzyme “constants” – – – – – • Reversible inhibition – • Km Vmax kcat kcat/Km Ki E+S+I ES + I EI ESI Impact on Km and Vmax for each Irreversible inhibition – I combines/binds to E to form a very stable complex E+P 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. Acylation Cleavage of peptide bond and formation of ester with enzyme 2. Deacylation Hydrolysis of ester and enzyme regenerated Kinetics → Mechanism Fast phase (burst phase/pre-steady state) Kinetics → Mechanism Histidine must be deprotonated for rxn to occur Ile (N-term) must be protonated for substrate to bind Chymotrypsin: “Catalytic triad” protease • Catalytic triad components – Nucleophile-Ser195 – His57 (general base) – Asp102 (stabilizes + charge on His) • Oxyanion hole – Stabilizes O- in tetrahedral intermediate 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 enzymesubstrate 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 K0.5 Change 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 1phosphate 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