PHA111 Enzyme Kinetics.pptx

MPharm Programme Enzyme Kinetics Dr. Mark Gray Slide 1 of 38 MPharm PHA111 Enzyme Catalysis Recap: Enzyme Catalysis Slide 2 of 38 MPharm PHA111 Enzyme Catalysis Steady State Kinetics • Pre-steady state: Isolated enzyme mixed with substrate to build-up an enzyme-substrate complex. Steady state...

MPharm Programme Enzyme Kinetics Dr. Mark Gray Slide 1 of 38 MPharm PHA111 Enzyme Catalysis Recap: Enzyme Catalysis Slide 2 of 38 MPharm PHA111 Enzyme Catalysis Steady State Kinetics • Pre-steady state: Isolated enzyme mixed with substrate to build-up an enzyme-substrate complex. Steady state is reached in ms. • Enzymes exist in the steady state most of the time to regulate metabolism. • new material produced = current material destroyed. Slide 3 of 38 MPharm PHA111 Enzyme Catalysis 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 a [E]0 • Typically, look at first minute of reaction, already well into the steady state domain. Slide 4 of 38 MPharm PHA111 Enzyme Catalysis However, in the real world……. • V0 follows ‘saturation kinetics’ re: [S] • At low [S], V0 a [S] i.e. the reaction rate is linear re: [S]. • At very high [S], V0 approaches Vmax, • This is the theoretical maximum rate at which the enzyme can operate. V 0 = [E ]0 [S ]k c a t K M + [S ] Michealis-Menten Equation Slide 5 of 38 MPharm PHA111 Enzyme Catalysis Slide 6 of 38 MPharm PHA111 Enzyme Catalysis Slide 7 of 38 MPharm PHA111 Enzyme Catalysis KM is the Michaelis Constant Slide 8 of 38 MPharm PHA111 Enzyme Catalysis Basic ways of obtaining KM Slide 9 of 38 MPharm PHA111 Enzyme Catalysis The Michaelis-Menten Mechanism E+S Ks ES kcat 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 PHA111 Enzyme Catalysis From this mechanism…… E+S Ks ES kcat E+P Ks = [E][S] and V0 = kcat[ES] [ES] but [E]0 = [E] + [ES] Slide 11 of 38 MPharm PHA111 Enzyme Catalysis Plugging those in we get….. [ES] = [E]0[S] Ks + [S] V0 = [E]0[S]kcat Ks + [S] Slide 12 of 38 MPharm PHA111 Enzyme Catalysis That looks a bit familiar V0 = [E]0[S]kcat V0 = [E]0[S]kcat c.f. Ks + [S] 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 PHA111 Enzyme Catalysis Slide 14 of 38 MPharm PHA111 Enzyme Catalysis The Lineweaver-Burk Plot • More accurate way of determining Vmax and KM. • Double reciprocal graph of measured rate V0 vs substrate concentration [S]. KM 1 = V0 Vmax[S] + y = + c mx 1 Vmax Slide 15 of 38 MPharm PHA111 Enzyme Catalysis Disadvantages of Lineweaver-Burk • Compression of data points with high [S] into small region V0 • This favours data with low [S]. y • Eadie & Hofstee decides to multiply everything through by V0Vmax giving: Slide 16 of 38 PHA111 MPH117Enzyme Catalysis = Vmax - KMV0 [S] = - mx c The Eadie-Hofstee plot • Advantages: all values of [S] are weighted equally. • Generally considered to be more accurate determination of KM. • Disadvantages: time consuming. • Both axes depend on V0, introducing potential for experimental error. Vmax grad = -KM V0 Slide 17 of 38 MPharm PHA111 Enzyme Catalysis V0/[S] The Woolf-Hanes plot • Multiply terms for L-B plot through by [S]. • Advantage: quicker to obtain data than E-H. • Disadvantage, KM is intercept not slope, thus more prone to error than E-H. [S]/V0 -KM Slide 18 of 38 MPharm PHA111 Enzyme Catalysis grad = 1/Vmax KM/Vmax [S] Back to the M-M equation • Can we compare catalytic efficiencies of different enzymes? • Enzymes have different kcat and KM values → cellular environment. • By assuming that [S] << KM we can derive a new equation in the form of a 2nd Order reaction. Slide 19 of 38 MPharm PHA111 Enzyme Catalysis V0 = [E]0[S]kcat KM + [S] [S] << KM V0 = kcat[E]0[S] KM kcat/KM : the specificity constant Slide 20 of 38 MPharm PHA111 Enzyme Catalysis Many enzymes use more than one substrate Slide 21 of 38 MPharm PHA111 Enzyme Catalysis Dihydrofolate reductase NADP+ Tetrahydrofolate Slide 22 of 38 MPharm PHA111 Enzyme Catalysis Both substrates bound Slide 23 of 38 MPharm PHA111 Enzyme Catalysis Lineweaver-Burk behaviour of a ternary complex • Several different LB plots. • Each plot has [S2] held constant. • Lines intersect. Slide 24 of 38 MPharm PHA111 Enzyme Catalysis LB behaviour for a Ping-Pong mechanism. • Same experimen tal set-up. • Each LB plot is parallel to one another. Slide 25 of 38 MPharm PHA111 Enzyme Catalysis Many drugs act via Reversible Inhibition • Most common form of inhibition. • e.g use of ethanol to treat methanol poisoning. • Inhibits alcohol dehydrogenase catalysed formation of formaldehyde, preventing blindness. Slide 26 of 38 MPharm PHA111 Enzyme Catalysis • Inhibitor only binds to ES complex. • This stabilises the complex making it harder for the substrate to react and leave. • Uncompetitive inhibition never seen in isolation. Slide 27 of 38 MPharm PHA111 Enzyme Catalysis • Binds to E or ES. Slide 28 of 38 MPharm PHA111 Enzyme Catalysis Examples of Uncompetitive / Mixed Inhibition HO O H P N HO O OH • N-(phosphonomethyl)glycine, aka glyphosate, aka ‘Roundup’. • Uncompetitively Inhibits 3-phosphoshikimate 3caboxyvinyltransferase. • Lithium Ion treatment of manic depression → Uncompetitive Inhibition of myo-inositol monophosphatase Slide 29 of 38 MPharm PHA111 Enzyme Catalysis • a is a measure of the extent of change in Michaelis Constant (a=1 for no inhibition). • a′ measures changes in Vmax, which in turn will affect KM. Slide 30 of 38 MPharm PHA111 Enzyme Catalysis LB plot for Competitive inhibition • For each LB plot [E] and [I] are held constant. • [S] is then systematically varied and V0 determined. • For competitive inhibition Vmax will be unaffected. Slide 31 of 38 MPharm PHA111 Enzyme Catalysis Uncompetitive Inhibition • Same experimental set-up. • Increasing [I] results in parallel LB plots. • Both KM and VMAX are Slide 32 of 38 MPharm PHA111 Enzyme Catalysis altered. Mixed Inhibition • Same experimental set-up. • Increasing [I] results in crossing LB plots. • Both KM and VMAX are altered. Slide 33 of 38 MPharm PHA111 Enzyme Catalysis Irreversible Inhibition • Covalent bonding to or modification of active site. • Permanent blockage of catalytic activity. • Basis of nerveagent attack upon serine proteases. Slide 34 of 38 MPharm PHA111 Enzyme Catalysis LB plot of Irreversible inhibition. • As inhibitor is added active [E] drops. • This will decrease VMAX but leave KM unchanged. • A rare form of reversible inhibition termed noncompetitive inhibition displays the same behaviour. 1/[V0] [I] -1/KM Slide 35 of 38 MPharm PHA111 Enzyme Catalysis 1/VMAX 1/[S] Other examples of Irreversible inhibition • Arsenic • Heavy metals • Alkylating Agents • Cyanide Ion Slide 36 of 38 MPharm PHA111 Enzyme Catalysis Enzyme-SH Metalloproteins Non-competitive Inhibitors • Bind to allosteric sites rather than the active site. • Act as enzyme switches. • Example: NonNucleoside Reverse Transcriptase Inhibitors (NNRTIs)→HIV1. Slide 37 of 38 MPharm PHA111 Enzyme Catalysis N N N N O Nevirapine H H N Cl O O CF3 Efavirenz Summary • The Michaelis Constant and the ‘turnover number’ are important descriptors of enzyme efficiency. • The catalytic activity of enzymes can be modified in distinctive ways by the addition of inhibitors. • These can be reversible or irreversible. • Reversible inhibitors come in several varieties. • The type of inhibition displayed can be determined via enzyme kinetics. Slide 38 of 38 MPharm PHA111 Enzyme Catalysis