Human Biochemistry, Volume 1 - Murray R. 1993

Structure and Function of Proteins and Enzymes
Enzymes: Kinetics
Substrate Concentration

In the following Structure/133.html">Discussion, we will consider Enzymatic reactions involving a single substrate and a single product. While this scenario is indeed observed in some enzymatic reactions, most of them involve two or more substrates and products. This, however, in no way diminishes the value of the subsequent reasoning. What holds true for a single substrate remains valid for two as well.

As the substrate concentration [S] increases while all other conditions are kept constant, the initial velocity V (the rate measured during the period when only a very small fraction of the substrate has been consumed) will rise to a maximum value V_max, after which it remains constant (Fig. 8.14).

As the substrate concentration increases, the rate will rise until the enzyme becomes saturated with substrate. Under these conditions, the measured initial velocity will no longer increase with a further increase in substrate concentration. Note that the substrate is typically present in a substantial molar excess relative to the enzyme. For example, if an enzyme with a Molecular Weight of 100,000 interacts with a substrate with a molecular weight of 100, and both are present at a concentration of 1 mg/ml, there will be 1,000 moles of substrate for every mole of enzyme. More realistic values are as follows:

Class="center">[Enz] = 0.1 µg/ml = 10^-9 M,

[S] = 0.1 mg/ml = 10^-3 M,

i.e., the molar excess of substrate relative to the enzyme is 10⁶.

Fig. 8.14. Effect of Substrate concentration on The rate of an enzymatic reaction.

Even if [S] is reduced 100-fold, its concentration will still exceed the Enzyme Concentration by 10,000-fold.

The situation corresponding to points A, B, and C in Fig. 8.14 is illustrated in Fig. 8.15. At points A and B, only a fraction of the enzyme molecules are complexed with the substrate, even though there are far more substrate molecules than enzyme molecules. This occurs because the Equilibrium Constant for the reaction Enz + S Enz — S (Formation of the Enz — S complex), while large, is finite. Thus, at points A and B, increasing or decreasing [S] will lead to an increase or decrease in the fraction of Enz molecules bound to S (i.e., the fraction of Enz — S molecules), and v will depend on [S]. At point C, virtually all enzyme molecules are bound to the substrate, and a further increase in [S], although increasing the frequency of collisions between Enz and S, cannot increase the reaction rate, as there are no longer any free enzyme molecules available to react with the substrate.

Case B is of particular theoretical interest because, in this case, exactly half of the enzyme molecules are saturated with substrate. Consequently, the velocity is equal to half of the maximum velocity (V_mах/2) achievable at the given enzyme concentration.