THE SIGNIFICANCE OF Km, kcat, and kcat /Km As described in the text, Km is an approximate measure of substrate affnity for the enzyme: it is numerically equal to the concentration of [S] at V = 0.5 Vmax. In general, a lower value of Km means tighter substrate binding. In fact, for those cases where kcat is much smaller than k–1, the Km will be equal to Kd, the dissociation constant for substrate binding to the enzyme (Kd = 1/Ka; see Figure 3–44). We have seen that k cat is the turnover number for the enzyme. At very low substrate concentrations, where 20 0 0 2 4 6 8 40 60 80 [S] mmole/liter To obtain Vmax and Km from such data, a double-reciprocal [S] << Km, most of the enzyme is free. Thus we can think of [E] = [Eo], so that the Michaelis–Menten equation becomes V = kcat/Km [E][S]. Thus, the ratio kcat/Km is equivalent to the rate constant for the reaction between free enzyme and free substrate. A comparison of kcat/Km for the same enzyme with different substrates, or for two enzymes with their different substrates, is widely used as a measure of enzyme effectiveness.   For simplicity, in this Panel we have discussed enzymes that have only one substrate, such as the lysozyme enzyme described in the text (see p. 144). Most enzymes have two substrates, one of which is often an active carrier
SOME ENZYMES ARE DIFFUSION LIMITED The values of kcat, Km, and kcat /Km for some selected molecule—such as NADH or ATP.   A similar, but more complex, analysis is used to determine the kinetics of such enzymes—allowing the order of substrate binding and the presence of covalent intermediates along the pathway to be revealed.
plot is often used, in which the Michaelis–Menten equation has merely been rearranged, so that 1/V can be plotted versus 1/ [S]. 1/V= + 1/ Vmax Km Vmax [S] 1 123468 1 Vmax 0.01 0.02 0.03 0.04 slope = KM / Vmax 1/V (second/µmole) enzymes are given below: Because an enzyme and its substrate must collide before they can react, kcat /Km has a maximum possible value that is limited by collision rates. If every collision forms an enzyme–substrate complex, one can calculate from diffusion theory that kcat /Km will be between 108 and 109 sec–1M–1, in the case where all subsequent steps proceed immediately. – 0.25 0 0.25 0.5 0.75 1.0– 0.5 fumarase fumarate 8x102 5x10–6 1.6x108 catalase H2O2 4x107 1 4x107 acetylcholinesterase acetylcholine 1.4x104 9x10–5 1.6x108 enzyme substrate kcat (sec–1) kcat/Km (sec–1M–1) Km (M)
Thus, it is claimed that enzymes like acetylcholinesterase and1 liter/mmole fumarase are “perfect enzymes,” each enzyme having evolved to the point where nearly every collision with its substrate converts the substrate to a product. [S] Km