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Thus, when the substrate concentration is very large, equation 15.9 reduces to

v 0 ¼kþ 2 [E], i.e. the initial rate is directly proportional to the enzyme concentration.

This is the basis of the experimental determination of enzyme activity in a particular

biological sample (Section 15.3). Figure 15.3 illustrates the importance of the correct

measurement of initial rate.

15.2.2 Inhibition of monomeric enzyme reactions

Competitive reversible inhibition

Reversible inhibitorscombine non-covalently with the enzyme and can therefore be

readily removed by dialysis.Competitive reversible inhibitorscombine at the same

site as the substrate and must therefore be structurally related to the substrate. An

example is the inhibition of succinate dehydrogenase by malonate:

CH 2 COOH

COOH

malonic acid (inhibitor)

no reaction

CH 2 COOH

CHCOOH



CHCOOH

succinic acid (substrate)

fumaric acid (product)

succinate dehydrogenase

CH 2 COOH

̄ ̄

All types of reversible inhibitors are characterised by their dissociation constantKi,

called theinhibitor constant,which may relate to the dissociation of EI (KEI)orofESI

(KESI). For competitive inhibition the following two equations can be written:

EþS ! ES! EþP

EþI ! EI!no reaction

Since the binding of both substrate and inhibitor involves the same site, the effect of a

competitive reversible inhibitor can be overcome by increasing the substrate concen-

tration. The result is thatVmaxis unaltered but the concentration of substrate required

to achieve it is increased so that when 0 ¼0.5Vmaxthen:

½SŠ¼Km 1 þ½IŠ

Ki



ð 15 : 10 Þ

where [I] is the concentration of inhibitor.

It can be seen from equation 15.10 thatKiis equal to the concentration of inhibitor

that apparently doubles the value ofKm. With this type of inhibition,Kiis equal toKEI

whilstKESIis infinite because no ESI is formed. In the presence of a competitive

inhibitor, the Lineweaver–Burk equation (15.5) becomes:

1

 0 ¼

Km

Vmax

1

½SŠ^1 þ

½IŠ

Ki



þ

1

Vmax ð^15 :^11 Þ

Application of this equation allows the diagnosis of competitive inhibition

(Fig. 15.5a). The numerical value ofKican be calculated from Lineweaver–Burk plots

for the uninhibited and inhibited reactions. In practice, however, a more accurate

592 Enzymes