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Michaelis-Menten kinetics

Enzymes catalyze reactions in physiological systems. In an equilibrium, an enzyme (E) binds a substrate (S) to form an enzyme-substrate complex (E-S). The E-S complex can dissociate or irreversibly convert the substrate to a product (P) (Scheme 1). The Michaelis-Menten equation describes the relationship between the rate of substrate conversion by an enzyme to the concentration of the substrate (Equation 1). In this equation, V is the rate of conversion, Vmax is the maximum rate of conversion, [S] is the substrate concentration, and Km is the Michaelis constant. The Michaelis constant is equivalent to the substrate concentration at which the rate of conversion is half of Vmax. Km approximates the affinity of enzyme for the substrate. A small Km indicates high affinity, and a substrate with a smaller Km will approach Vmax more quickly. Very high [S] values are required to approach Vmax, which is reached only when [S] is high enough to saturate the enzyme. While the derivation is not shown in this discussion, Vmax is equivalent to the product of the catalyst rate constant (kcat) and the concentration of the enzyme.

Scheme 1 - enzyme-substrate complex and product formation model

(1) Applet This applet plots V vs [S] data for up to three compounds/situations. For each compound, Km and Vmax must be specified. The units of Km and [S] are concentration, e.g. mM or µM. The units of Vmax and V are amount of product over time, typically µmol/min or similar. Substrate (+ Inhibitor) (Color) one (blue) two (red) three (green) calculation may be slow Vmax (amount time-1)
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Km (conc.)
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One way is to look at the enzyme's kinetic behavior -. connecting together when they should be separate. Nathanael Gray and colleagues screened for new Bcr-abl inhibitors by looking for small molecules that were toxic to Bcr-abl�expressing cells but not normal cells. The resulting fusion protein causes cellular signaling pathways to be on when they should be off and contributes to the development of CML. For this model.102 A new inhibitor of Bcr-abl kinase. CML is a common form of leukemia that is typically characterized by two genes. The highly selective Bcr-abl inhibition and distinct mode of action suggest that this inhibitor is a valuable lead for developing new CML treatments. so there is a strong need to find therapeutics with alternative mechanisms of action. The latter stands for the appearance of the product P in solution (+ d[P]/dt) whose phenomenological rate equation (first-order) is given by v0 = kcat[ES] --------(2). A compound identified in the screen was shown to selectively inhibit leukemic cell lines and use a different mechanism to bind to the protein from the current inhibitor. CML patients are developing resistance to the current clinical inhibitor of these signaling pathways. is reported in the February issue of Nature Chemical Biology. If a newly discovered enzyme obeys that rate law. So we want to know what rate law such an enzyme would obey. BCR and ABL1. We would like to know how to recognize an enzyme that behaves according to this model. How to derived Michael-Menten A simple model of enzyme action: In this model. . then we can assume that it acts according to this model.at how substrate concentration affects its rate. let v0 be the initial velocity of the reaction. and some of the resulting complex ES is allowed to break down and yield the product P and the free enzyme back. Let's derive a rate law from this model.How km inhibit leukemia pp 95 . the substrate S reversibly associates with the enzyme E in a first step. a key therapeutic target for treating chronic myelogenous leukemia (CML).

the rate of formation of [ES] (one second order kinetic step) equals its rate of consumption (two first order kinetic steps). During the initial phase of the reaction. like [S] and [E]total . the reaction is in a steady state. so that we can replace it in (2). Before proceeding.v0. During this phase. These assumptions. as long as the reaction velocity remains constant. thus [P] << [S]). while that for the enzyme is [E]total = [E]free + [ES] (the possible formation of a complex EP is not considered here). k-1[ES] + kcat[ES] = k1[E][S] (3) Remember that we are trying to solve for [ES] in terms of measurables. with ES being formed and consumed at the same rate. So we must replace the unknown [ES] in (2) with measurables. a kinetic parameter kcat. one should state (and remember) some implicite assumptions: • As long as initial velocity is considered. According to model (1). So in the steady state. • We want to express v0 in terms of measurable (experimentally defined.[ES]. Rate of consumption of [ES] = k-1[ES] + kcat [ES]. and The concentration of substrate is in large excess over that of the enzyme ([E] << [S]). so we can see how to test the mechanism by experiments in kinetics. independent) variables. and another variable unknown to us . First. and the concentrations (variables) in (3): . collect the kinetic constants. Rate of formation of [ES] = k1[E][S]. the concentration of product can be neglected (compared to that of the substrate. which hold in most kinetic experiments performed in test tubes at low enzyme concentration. are convenient when considering the mass conservation equations for the reactants [S]0 = [S]free + [ES] + [P] which now approximates to [S]0 = [S].containing an experimentally measurable (dependent) variable .

and (k-1 + kcat)/k1 = [E][S]/[ES] To simplify (4).): [ES] Km = [E]total[S] . to limit the number of unknowns: [E] = [E]total .[ES][S] Then collect terms containing [ES] on the left: [ES] Km + [ES][S] = [E]total[S] Factor [ES] from the left-hand terms: [ES](Km + [S]) = [E]total[S] and finally.. first group the kinetic constants by defining them as Km: Km = (k-1 + kcat)/k1 (5) (4) and then express [E] in terms of [ES] and [E]total. divide both sides by (Km + [S]): (7) (6) [ES] = [E]total [S]/(Km + [S]) (8) .(k-1 + kcat) [ES] = k1 [E][S].[ES]) [S]/[ES] Solve (7) for [ES]: First multiply both sides by [ES] (no Black Magic involvement here.[ES] Substitute (5) and (6) into (4): Km = ([E]total ..

a low value for Km may indicate a high affinity of the enzyme for its substrate. Thus. Another physically meaningful limit of this function is found at vanishingly small values of [S] (--> 0).Km. describes the kinetic behavior of an enzyme that acts according to the simple model (1). has no physical meaning (no negative concentrations). * the second parallel to the v0 axis at [S] = . the Michaelis-Menten equation. Equation (11). just like the saturation equation for the binding of oxygen to myoglobin. Further analysis reveals the physical meaning of Km: the concentration of substrate at which the velocity is half Vmax. Equation (11) is of the form y = ax/(b + x) (does this look familiar?) This is the equation of a rectangular hyperbola.that is. will be different for every enzyme-substrate pair. Mathematically. . the initial substrate concentration. when all enzyme molecules are tied up with S. The two kinetic parameters.Substitute (8) into (2): v0 = kcat[E]total [S]/(Km + [S]) (9) The maximum velocity Vmax occurs when the enzyme is saturated -. represents the velocity at infinite [S] (saturation). substituting Km for [S] in (11) yields v0 = 1/2 Vmax. Indeed. Thus. Vmax and Km . Vmax = kcat [E]total Substitute Vmax into (9) for kcat [E]total: v0 = Vmax [S]/(Km + [S]) (11) (10) This equation expresses the initial rate of reaction in terms of a measurable quantity. the function v0 presents two asymptotes: * one parallel to the [S] axis at v0 = Vmax . or [ES] = [E]total.

a plot of v0 versus [S] will be a rectangular hyperbola. kcat ) * the effectiveness of productive substrate binding (affinity. often referred to as the catalytic ability of the enzyme. In this example. reciprocal plot: 1/velocity vs. sometimes referred to as a MichaelisMenten plot. displaying pseudo-first order kinetics in [S]. kcat /Km recombines the two traditionally-separated aspects of enzyme catalysis: * the effectiveness of transformation of bound product (catalysis per se. Relationship between Michaelis-Menten and Lineweaver-Burk plot. 1/Km = k1 /(k-1 + kcat)) Equation (11) means that. unless we find other evidence to the contrary. In this case. we assume that they act according to model (1). with an inset Lineweaver-Burk plot. the velocity becomes proportional to the (low. is a direct measure of the efficiency of the enzyme in transforming the substrate S. The parameter Vmax /Km (or rather its constant part kcat /Km). substrate concentration.where v0 --> Vmax /Km [S]. Lineweaver-Burk analysis is one method of linearizing substrate-velocity data so as to determine the kinetic constants Km and Vmax. When enzymes exhibit this kinetic behavior. for an enzyme acting according to the simple model (1). and call them Michaelis-Menten enzymes. One creates a secondary. we'll make a combination graph commonly used to characterize enzyme activitya curve of initial velocity vs. 1/ . relative to Km) substrate concentration.

Yet the Lineweaver-Burk plot continues to be a useful visual tool. X intercept = -1/Km slope = Km/Vmax Now that programs such as Prism easily do nonlinear regression. the best way to determine Km and Vmax is to fit a hyperbola directly to the substrate-velocity data.[substrate]. so that it reflects the best possible estimates of Kd and Bmax. These enzymes are so efficient they effectively catalyse a reaction each time they encounter a substrate molecule and have thus reached an upper theoretical limit for efficiency (diffusion limit).[10] . the Lineweaver-Burk plot is a straight line with Y intercept = 1/Vmax. but we'll superimpose a line based upon nonlinear regression analysis. these enzymes have often been termed perfect enzymes. Range The most efficient enzymes reach a k2 / KM in the range of 108 .1010 M−1 s−1. When catalytic activity follows Michaelis-Menten kinetics over the range of substrate concentrations tested. particularly because of its characteristic shifts in the presence of various types of inhibitors. So we'll create a Lineweaver-Burk plot with data points derived from double-reciprocal transformation.