Enzyme Kinetics Lecture Slides PDF

MPharm Programme Enzyme Kinetics Professor Mark Gray Slide 1 of 38 MPharm PHA114 Enzyme Kinetics  Recap: Enzyme Catalysis Slide 2 of 38 MPharm PHA114 Enzyme Kinetics  Steady State Kinet...

MPharm Programme Enzyme Kinetics Professor Mark Gray Slide 1 of 38 MPharm PHA114 Enzyme Kinetics  Recap: Enzyme Catalysis Slide 2 of 38 MPharm PHA114 Enzyme Kinetics  Steady State Kinetics Pre-steady state: Isolated enzyme mixed with substrate to build-up an enzyme-substrate complex. Steady state is reached in s. Enzymes exist in the steady state most of the time to regulate metabolism. new material produced = current material destroyed. Slide 3 of 38 MPharm PHA114 Enzyme Kinetics  Some assumptions….. Concentration of Enzyme ([E]) is negligible compared to substrate ([S]). Examine initial reaction rate V0: no depletion in substrate pool, no accumulation in products. This would mean that V0  [E]0 Typically, look at first minute of reaction, already well into the steady state domain. Slide 4 of 38 MPharm PHA114 Enzyme Kinetics  However, in the real world……. V0 follows ‘saturation kinetics’ re: [S] At low [S], V0  [S] V0 = [E]0[S]kcat i.e. the reaction rate is linear re: [S]. KM + [S] At very high [S], V0 approaches Vmax, Michealis-Menten This is the theoretical Equation maximum rate at which the enzyme can operate. Slide 5 of 38 MPharm PHA114 Enzyme Kinetics Slide 6 of 38 MPharm PHA114 Enzyme Kinetics Slide 7 of 38 MPharm PHA114 Enzyme Kinetics  KM is the Michaelis Constant Slide 8 of 38 MPharm PHA114 Enzyme Kinetics  Basic ways of obtaining KM Slide 9 of 38 MPharm PHA114 Enzyme Kinetics  The Michaelis-Menten Mechanism Ks kcat E+S ES E+P Formation of ES is rapid and reversible. ES is a non-covalent complex: i.e. all the ‘chemistry’ takes place in the 2nd step. kcat is known as the turnover number. This step will follow 1st order kinetics. Slide 10 of 38 MPharm PHA114 Enzyme Kinetics  From this mechanism…… Ks kcat E+S ES E+P Ks = [E][S] and V0 = kcat[ES] [ES] but [E]0 = [E] + [ES] Slide 11 of 38 MPharm PHA114 Enzyme Kinetics  Plugging those in we get….. [ES] = [E]0[S] Ks + [S] V0 = [E]0[S]kcat Ks + [S] Slide 12 of 38 MPharm PHA114 Enzyme Kinetics  That looks a bit familiar V0 = [E]0[S]kcat V0 = [E]0[S]kcat Ks + [S] c.f. KM + [S] Often KM is very close to KS, meaning that M-M kinetics are operable. KM > KS : dissociation of ES is significant in comparison to kcat (Briggs-Haldane Kinetics). KM < KS : long-lived intermediates exist after substrate binding. Slide 13 of 38 MPharm PHA114 Enzyme Kinetics Slide 14 of 38 MPharm PHA114 Enzyme Kinetics  The Lineweaver-Burk Plot More accurate way of determining Vmax and KM. Double reciprocal graph of measured rate V0 vs substrate concentration [S]. 1 KM 1 = + V0 Vmax[S] Vmax y = mx + c Slide 15 of 38 MPharm PHA114 Enzyme Kinetics  Disadvantages of Lineweaver-Burk Compression of data points with high [S] into small region K MV 0 This favours data V0 = Vmax - [S] with low [S]. Eadie & Hofstee y = c - mx decides to multiply everything through by V0Vmax giving: Slide 16 of 38 PHA111 PHA114 Enzyme Kinetics  The Eadie-Hofstee plot Advantages: all values of [S] are weighted equally. Vmax Generally considered to be more accurate grad = -KM determination of KM. Disadvantages: time V0 consuming. Both axes depend on V0, introducing potential for experimental error. V0/[S] Slide 17 of 38 MPharm PHA114 Enzyme Kinetics  The Woolf-Hanes plot Multiply terms for L-B plot through [S]/V0 by [S]. grad = Advantage: 1/Vmax quicker to obtain data than E-H. Disadvantage, KM -KM is intercept not slope, thus more KM/Vmax prone to error than E-H. [S] Slide 18 of 38 MPharm PHA114 Enzyme Kinetics  Back to the M-M equation Can we compare catalytic efficiencies of different V0 = [E]0[S]kcat enzymes? KM + [S] Enzymes have different kcat and KM values → [S]