multi-substrate enzyme kinetics

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Multi-Substrate Enzyme Kinetics. Kinetics of Multi-Substrate Enzymes * Single substrate/product enzyme-catalyzed reactions of the type we’ve been studying, i.e. S P are useful for gleaning kinetic principles, but are actually very rare. - PowerPoint PPT Presentation

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  • Multi-Substrate Enzyme Kinetics

  • Kinetics of Multi-Substrate Enzymes

    * Single substrate/product enzyme-catalyzed reactions of the type weve beenstudying, i.e. S P

    are 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 + Q

    so 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).

  • Terminology

    Substrates:A, B, C, DProducts:P, Q, R, SEnzymes:E, F, GWith 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 TerQuad

  • Kinetic Mechanisms

    The sequence in which substrates are bound and products are released.

    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 + Q

  • Ordered Sequential Mechanism

    Substrates bind to the enzyme in a defined sequence, and products are released in a defined sequence. E + A EAEA + B (EAB) --central complex

    The enzyme-substrate central complex is then converted to enzyme-product central complex: (EAB) (EPQ)

    The products are now released:

    (EPQ) EQ + PEQ E + Q

    We can represent this schematically in a Cleland Plot:

    Ordered Sequential BiBikinetic mechanism(note parentheses are sometimesleft off of the central complexes)

  • 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 + PF = 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 + Q

  • Ping-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 Systems

    For 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 if

    A = variable substratethen a plot of v vs. [A] will give a hyperbolic Michaelis-B = fixed substrateMenten 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 enzyme

    A + B P + Q

    at 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 the fixed substrate would have on the velocity at very high concentrations of the variable substrate. -- A change in slope indicates the effect that a change in concentration of the fixed substrate would have on the velocity at very low concentrations of the variable substrate. Remember that the 1/slope of LB plot is the rate constant at low substrate concentrations (V/Km).

  • Substrate Concentration Assays with Sequential Enzymes

    Consider a BiBi ordered sequential reaction: A + B P + Q

    The Cleland plot for such a reaction would be:

    We'll discuss the results of a set of enzyme assays in which substrate A is used as the variable substrate and substrate B as the fixed substrate.

    At very low [A]: -- the rate limiting step of the reaction would be the binding of A to the enzyme as the substrate is in very short supply.-- an increase in the concentration of B would reduce the concentration of the EA complex in the reaction mixture by binding to it to form EAB complex. Reducing the concentration of the product of the E + A EA reaction will pull it to the right by the Law of Mass Action. So the increase in B has increased the speed of the rate limiting step, and therefore of the overall reaction. As we're looking at effects at low A concentrations this would be seen as a change in the slope of the Lineweaver-Burk plot.

    At very high [A]:-- the rate limiting step would be the EAB EPQ, which is the inherently slowest reaction, or EA + B EAB at low B levels.-- an increase in B would increase the speed of either of these as a reactant in the second reaction and by the effect of generating more EAB for the central reaction to occur. This would be seen as a change in V as we are considering the effects at high A concentration.

  • A similar result would be obtained if the assay was reversed and B used as the variable substrate or if the enzyme used a random sequential mechanism.Ordered Sequential Reaction:

    Change in concentration of fixed substratebrings about a change in both the slopeand intercept of Lineweaver-Burk plots.

  • Substrate Concentration Assays with Ping-Pong Enzymes

    Consider a BiBi ping-pong reaction: A + B P + Q

    The Cleland plot for such a reaction would be:

    We'll discuss the results of a set of enzyme assays in which substrate A is used as the variable substrate and substrate B as the fixed substrate.

    The critical difference about a ping-pong reaction is that a product leaves the enzyme before all the substrates have bound. This is going to change the way in which the substrates interact with each other, kinetically. Remember that in normal enzyme assays we are measuring the initial velocity - i.e. the rate immediately after addition of enzyme to the substrates. Under these conditions the concentration of products is zero - no product has had time to be produced yet - and the reaction:

    FP F + P

    which is normally a readily reversible reaction, is effectively irreversible since the back reaction would require a supply of product P. We'll see how this affects the kinetics of the reaction when we examine the influence of B, as the fixed substrate, at low concentrations of A.

  • Effects of Substrate Concentration on Ping-Pong Kinetics, Contd

    At very high [A]:-- V increases with increasing concentration of fixed substrate as with sequential, so intercept of LB plot decreases.

    At very low [A]: -- Like sequential, at low [A] the reaction E + A EA will be rate limiting.-- Increasing the [B] will convert more F to FB and thereby decrease the concentration of enzyme form F. At this point you might expect the Law of Mass Action to take over. The reduction of F would pull the FP F + P reaction to the right reducing the concentration of FP which, in turn, would pull EA FP to the right which would have the same effect on the rate limiting step.

    *** But remember that FP F + P is effectively irreversible due to initial velocities!*** -- The reduction in F would seem to pull this to the right because the loss of F would decrease the speed of the back reaction. However it has no direct effect on the speed of the

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