Enzyme Kinetics-II(Detail)

Michaelis-Menten Equation Study of the impact made on the rate of an enzyme-catalysed reaction by changes in experimental conditions is known as enzyme kinetics. Knowledge of kinetics can be a very useful tool in understanding the mechanism by which an enzyme carries out its catalytic activity. The effect of substrate concentration on the initial rate of an enzymecatalysed reaction is a central concept in enzyme kinetics. When data are generated from experiments of this type and the results plotted as a graph of initial rate (v, y-axis) against substrate concentration ([S] *, x-axis), many enzymes exhibit a rectangular hyperbolic curve like the one shown in the diagram.

Observations of this type lead Leonor Michaelis and Maud Menten to think about the possible reason why a curve should follow this shape and led them to derive an algebraic equation that now bears their names. There are several modern ways to explain the way in which the Michaelis-Menten equation is derived. Deriving the Michaelis-Menten Equation Start with the generalised scheme for enzyme-catalysed production of a product (P) from substrate (S). The enzyme (E) does not magically convert S

into P, it must first come into physical contact with it, i.e. E binds S to form an enzyme-substrate complex (ES). Michaelis and Menten therefore set out the following scheme:

The terms k1, k-1 and k2 are rate constants for, respectively, the association of substrate and enzyme, the dissociation of unaltered substrate from the enzyme and the dissociation of product (= altered substrate) from the enzyme. Note that there is the theoretical possibility of a reverse reaction, with ES complex forming from E and P, but this can be ignored because we are considering initial rates of reaction, i.e. when the enzyme is first provided with substrate, so there should not be any product available to combine with enzyme. The overall rate of the reaction (v) is limited by the step ES to E + P, and this will depend on two factors - the rate of that step (i.e. k 2) and the concentration of enzyme that has substrate bound, i.e. [ES]. This can be written as:
(Equation 1)

At this point it is important to take note on two assumptions that are made in this scheme. The first is the availability of a vast excess of substrate, so that [S]>>[E]. Secondly, it is assumed that the system is in steady-state, i.e. that the ES complex is being formed and broken down at the same rate, so that overall [ES] is constant. The formation of ES will depend on the rate constant K1 and the availability of enzyme and substrate, i.e. [E] and [S]. The breakdown of [ES] can occur in two ways, either the conversion of substrate to product or the non-reactive dissociation of substrate from the complex. In both instances the [ES] will be significant. Thus, at steady state we can write:

The next couple of steps are rearrangements of this equation. First of all the rate constants can collected together on the right-hand side because they are both multiplied by [ES], this gives the following equation:

Then dividing both sides by (k-1 + k2), this becomes:

The three rate constants are noted now on the same side of the equation. As the name implies, these terms are constants, so they can actually be combined into one term. This new constant is termed the Michaelis constant and is written KM.

It is to be noted that the three rate constants in the definition of K M are actually inverted (the other way up) compared with our previous equation. Substituting this definition of KM into the previous equation now gives the following equation:

(Equation 2)

The total amount of enzyme in the system should be the same throughout the experiment, but it can either be free (unbound) E or in complex with substrate, ES. If the total enzyme is termed as E 0, this relationship can be written as follows:

This can be rearranged (by subtracting [ES] from each side) to give:

So, the [E] free in solution is equal to the total amount of enzyme minus the amount that has substrate bound. Substituting this definition of [E] back into equation 2 will give following equation:

This can now be rearranged in several steps. First of all, the bracket should be open so that the terms [E0] and [ES] are separately multiplied by [S]

Next, each side should be multiplied by KM, this give the following equation:

Then the two [ES] terms are collected together on the same side. This gives:

Then because both terms on the right-hand side are multiplied by [ES], they are collected together into a bracket:

Dividing both sides by (KM + [S]) now gives the following equation:

Substituting this left-hand side into Equation 1 in place of [ES] results in:

The maximum rate, which may be called as Vmax, would be achieved when all of the enzyme molecules have substrate bound. Under conditions when [S] is much greater than [E], it is fair to assume that all E will be in the form ES. Therefore [E0] = [ES]. Thinking again about Equation 1, the term Vmax could be substituted for v and [E0] for [ES]. This would give us:

It is to be noted that k2 [E0], which was present in the previous equation, so this could be replaced with Vmax, giving a final equation:

This final equation is actually called the Michaelis-Menten equation. Perhaps this derivation still leaves one puzzled about the importance of the Michaelis-Menten equation. The significance becomes clearer when one consider the case when the rate of reaction (v) is exactly half of the maximal reaction rate (Vmax). Under those circumstances, the Michaelis-Menten equation could be written:

On dividing both sides by Vmax this becomes:

Multiplying both sides by (KM + [S]) gives:

And then multiplying both sides by 2 further resolves the equation to:

2[S] on the right-hand side is the same as [S] + [S], so [S] could be taken out from each side. Thus when the rate of the reaction is half of the maximum rate:

The KM of an enzyme is therefore the substrate concentration at which the reaction occurs at half of the maximum rate. So the previous graph could be written as follows:

Conclusion KM is an indicator of the affinity that an enzyme has for a given substrate, and hence the stability of the enzyme-substrate complex.

After looking at the shape of the graph it is to be noted that at low [S], it is the availability of substrate that is the limiting factor. Therefore as more substrate is added there is a rapid increase in the initial rate of the reaction so any substrate is rapidly mopped up and converted to product. At the KM, 50% of active sites have substrate bound. At higher [S], a point is reached (at least theoretically) where all of the enzyme has substrate bound and is
working flat out. Adding more substrate will not increase the rate of the reaction, hence the levelling out observed in the graph.

There are limitations in the quantitative (i.e. numerical) interpretation of this type of graph, known as a Michaelis plot. The Vmax is never really reached and therefore Vmax and hence KM values calculated from this graph are somewhat approximate. A more accurate way to determine Vmax and KM (though still not perfect) is to convert the data into a linear Lineweaver-Burk plot. Let us examine enzyme kinetics as a function of the concentration of substrate available to the enzyme. A series of tubes were set up containing graded concentrations of substrate, [S]. At time zero, a fixed amount of the enzyme preparation is added. Over the next few minutes, the concentration of product formed was measured. If the product absorbs light, it can easily done in a spectrophotometer. Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of Vi. Plotting Vi as a function of [S], we find that -----1. At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S]. But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola). 2. The asymptote represents the maximum velocity of the reaction, designated Vmax. 3. The substrate concentration that produces a Vi that is one-half of Vm ax is designated the MichaelisMenten constant, Km(named after the scientists who developed the study of enzyme kinetics).

Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate). Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km. Vmax is determined by the point where the line crosses the 1/Vi = 0 axis (so the [S] is infinite). Note that the magnitude represented by the data points in this plot decrease from lower left to upper right. Km equals Vmax times the slope of line. This is easily determined from the intercept on the X axis.