南京大学-杨荣武生物化学-英文版-第二篇-EnzymeIII.ppt

Enzymes III,Enzyme Kinetics - Inhibition,Enzyme Inhibition,Enzyme can be inhibited by inhibitors. Inhibitors are tools to scientists to understand enzymes. Inhibitors are also in many cases pharmaceutical reagents against diseases;Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either reversible or irreversible;Reversible inhibitors associate with enzymes through non-covalent interactions. Reversible inhibitors include three kinds:Competitive inhibitors;Non-competitive inhibitors;Un-competitive inhibitors Irreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.,Competitve Inhibitors,Competitve Inhibitors,Often “resemble” substrates or cofactorsBind to enzyme through similar complementary interactionsExample: Dihydrofolate reductase,An example,HIV protease needs to bind and cleave polypeptide during life cycle,Competitive Inhibitors,,v,,,,,[S],vmax,,,,Km,k3,k-3,Km increasesvmax unchanged,,+inhibitor,,,Km(1+[I]/KI),-1/(Km(1+[I]/KI)),,Competitive Inhibitors,,v,,,,,[S],vmax,,,,Km,,,1/[S],1/v,1/vmax,-1/Km,,Slope=Km/vmax,,,KI,Km increasesvmax unchanged,,+inhibitor,,,Km(1+[I]/KI),-1/(Km(1+[I]/KI)),,Slope= Km(1+[I]/KI)/vmax,Practical case: Methanol poisoning,A wealthy visitor is taken to the emergency room of Gulou Hospital, where he is diagnosed with methanol poisoning. If you are a medical student and asked what to do? How would you suggest treating this patient?,Noncompetitive Inhibitors,,k1,k-1,,k2,KI,Km unchangedvmax decreases,KI’,Uncompetitive Inhibitors,,k1,k-1,,k2,Km decreasesvmax decreasesSlope unchanged,+inhibitor,KI’,,,1/[S],1/v,1/vmax,-1/Km,,Slope=Km/vmax,,,,(1+ KI/[I])/Vmax,,Slope= Km/vmax,v,,,,[S],vmax,,,Km,Km/(1+ KI/[I]),,,Vmax/(1+KI/[I]),,,- (1+ KI/[I])/Km,,,,,Irreversible Inhibitor,Combines with or destroys an essential functional group on the enzyme (e.g. forms covalent bonds)Inhibit enzymes irreversibly3 different types:Group Specific Reagent: - inhibitor does not resemble substrateSubstrate Analogue:- inhibitor resembles substrateSuicide Inhibitors:- inhibitor resembles substrate, turns "dangerous" after processed by enzyme,Group Specific Reagent,Does not resemble substrateirreversibly inactivates enzyme by modifying an essential R group e.g. DIPF (potent nerve gas) blocks acetylcholinesterase, which is essential for proper neural transmission,active site serine,,Substrate Analogue,Resemble substrateBinds at enzyme active siteThen irreversibly modifies (or binds to) to the active sitee.g. TPCK forms covalent bond with the Histidine at the active site of Chymotrypsin (a protease secreted by the pancreas),Substrate Analogue,Suicide Inhibitor,Resemble substrateBinds at enzyme active site (not an "inhibitor" yet)Processed by enzyme via normal catalytic mechanismBecomes a chemically active intermediates that modifies and inactivates the enzyme irreversiblyGood candidate for drug due to minimal side effect,Penicillin is a suicide inhibitor,Summary of Classes of Inhibitors,Competitive inhibition - inhibitor (I) binds only to E, not to ES Noncompetitive inhibition - inhibitor (I) binds either to E and/or to ES Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition.Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition is mixed.,Sample Questions,What is the v/Vmax ratio when [S]=5Km?Draw a Lineweaver-Burk plot if Vmax=100 mmol/mL, Km=2mM.Draw the new lineweaver-Burk plot on the same plot as above if a competitive inhibitor is added. [I]=0.5 mM, KI=1mM.,。