PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Multi-Substrate Enzyme KineticsKinetics of Multi-Substrate Enzymes* Single substrate/product enzyme-catalyzed reactions of the type we’ve beenstudying, i.e. S <==> Pare useful for gleaning kinetic principles, but are actually very rare.* Most enzymes have two or more substrates and frequently multiple products, i.e.A + B <==> P + Qso the kinetics are more complicated than the 1-substrate Michaelis-Menten systems.* Multi-substrate kinetics can be simplified somewhat by holding one substrate constantand varying the other, but to really understand the enzyme mechanism you have to consider all of the substrates (and even the products).TerminologySubstrates: A, B, C, DProducts: P, Q, R, SEnzymes: E, F, G With multi-substrate enzymes you often have toinvoke more than one enzyme form; E alwaysrepresents free enzyme-- the form before anysubstrate is bound.Inhibitors: I, JTransitory Complexes: Like enzyme-substrate complex, but there are more types with a multi-substrate enzyme, i.e. for A + B <=> P + Q enzyme you could haveEA, EB, EAB and even EP, EQ, EPQNot all enzymes make all of these transitory complexes. Some can evenmake combined enzyme-substrate-product transitory complexes such asEAP, EBQCentral Complexes: These are transitory complexes that are full, i.e. all substrates are boundor all products are bound. For A + B <=> P + Q the central complexescould be the following (usually in parentheses):(EAB), (EPQ)Reactancy: Uni, Bi, Ter, Quadi.e., number of reactants in a particular direction. Therefore A + B <=> P + Q is BiBiand A + B + C <=> P + Q + R + S is TerQuadKinetic MechanismsThe sequence in which substrates are bound and products are released.(1) Sequential (a.k.a. Ternary Complex).All substrates bind to the enzyme before the first product is released.* Ordered --substrates & products bound and released in obligatory sequence* Random --no obligatory binding sequence(2) Ping-Pong (a.k.a. Double Displacement or Substituted-Enzyme)At least one product is released before all substrates have bound.We will study the distinctions between these kinetic mechanismsUsing a typical BiBi enzyme:A + B <==> P + QOrdered Sequential MechanismSubstrates bind to the enzyme in a defined sequence, and products are released in a defined sequence. E + A <=> EAEA + B <=> (EAB) --central complexThe enzyme-substrate central complex is then converted to enzyme-product central complex: (EAB) <=> (EPQ)The products are now released: (EPQ) <=> EQ + PEQ <=> E + QWe can represent this schematically in a Cleland Plot:Ordered Sequential BiBikinetic mechanism(note parentheses are sometimesleft off of the central complexes)Random Sequential MechanismSimilar to Ordered Sequential except there is no specified order in which substrates must bind or products must be released. E + A <=> EAEA + B <=> (EAB)or Central complex is IDENTICALE + B <=> EB by either pathEB + A <=> (EAB)The next step is the same as with Ordered Sequential mechanism: (EAB) <=> (EPQ)We now have a choice of sequences of product release: (EPQ) <=> EQ + PCentral complex is IDENTICAL EQ <=> E + Qby either path or(EPQ) <=> EP + QEP <=> E + PRandom SequentialCleland PlotRandom & Ordered mechanisms are similar.Why are some enzymes ordered?-- binding of first substrate causes conformational changethat is required for binding second substrate, or-- first substrate binds directly to second substrate.-- kind of like Uncompetitive inhibitor binding to ES complex.Ping-Pong (Double Displacement) Mechanism-- At least one product is released before all of the substrates have bound. -- Common. Examples include serine proteases & aminotransferases. The first substrate binds in the usual way, except that the EA complex in this case isactually a central complex. The active site is full because substrate A will be converted to product before the second substrate can bind: E + A <=> (EA)The next reaction is the key to the whole process: (EA) <=> (FP) In this reaction a part of the substrate has been removed from substrate A, converting it to product P. The removed section has become covalently bound to the enzyme to create a new form of the enzyme, enzyme F. The first product of the reaction is now released and the second substrate binds: (FP) <=> F + P F = covalent enzyme-adductF + B <=> (FB)Now the stored section of the first substrate is transferred to the second substrate to create the second product, which is then released: (FB) <=> (EQ)(EQ) <=> E + QPing-Pong Mechanism: The Movie Cleland Plot for Ping-Pong BiBi Mechanism: Remember--(EA), (FP), (FQ), & (EQ) are CENTRAL COMPLEXES Note: Ping-Pong is an ordered kinetic mechanism, of necessity!Effects of Substrate Concentration in Multi-Substrate SystemsFor a single-substrate enzyme, i.e. A <=> P, a kinetic experiment measuring v as a functionof [A] gives you a hyperbolic, Michaelis-Menten-type curve, which can be analyzed viaany of the kinetic plots discussed previously, such as Lineweaver-Burk, Hanes, etc.With a multi-substrate enzyme, i.e a typical BiBi enzyme: A + B <=> P + Qyou can get the same result by holding one substrate constant and varying the other, so ifA = variable substrate then a plot of v vs. [A] will give a hyperbolic Michaelis-B = fixed substrate Menten curve, and vice-versa. Analysis would be the sameas with a single-substrate system.Now consider what would happen if you repeat the experiment with an increased conc. of the fixed substrate, B in this case.-- reaction rate will be faster at any given conc. of variable substrate, A.-- kinetic parameters will change to reflect changes in velocity.-- if you did this at several fixed concentrations of B and plotted the datasets on a Lineweaver-Burk plot, you would get a series of lines.Here is a typical Lineweaver-Burk pattern obtained for a BiBi enzymeA + B <=> P + Qat different fixed concentrations of substrate B.The actual pattern of lines obtained will vary according to the way in which the enzyme interacts with the two substrates, and enables us to distinguish between sequential and ping-pong enzymes. In discussing graphs of this type we'll consider changes in V and in the slope of the line. -- A change in V indicates the effect that a change in the concentration of