Food Biochemistry and Food Processing

164 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

(Eisenthal and Cornish-Bowden 1974) are useful in
estimating the Michaelis-Menten kinetic parameters
and are the better plots for revealing deviations from
the reaction behavior.

Hill Plot

The Hill equation is a quantitative analysis measur-
ing non–Michaelis-Menten behavior of cooperativi-
ty (Hill 1910). In a case of completely cooperative
binding, an enzyme contains hbinding sites that are
all occupied simultaneously with a dissociation con-
stant K[E][S]h/[ESh], where his the Hill coeffi-
cient, which measures the degree of cooperativity. If

there is no cooperativity, h1; there is positive

cooperativity when h1, negative cooperativity

when h1. The rate value can be expressed as v

Vmax[S]h/(K
[S]h), and the Hill equation gives a

linear log(vi/Vmaxv) hlog[S] logK,when

log(v/Vmaxv) is plotted as a function of log[S]

with a slope of hand a yintercept of -logK(Fig.

7.7). However, the equation will show deviations

from linearity when outside a limited range of sub-

strate concentrations, that is, in the range of [S] K.

FACTORS AFFECTING ENZYME

ACTIVITY

As discussed earlier in this chapter, the rate of an

enzymatic reaction is very sensitive to reaction con-

ditions such as temperature, pH, ionic strength, buf-

fer constitution, and substrate concentration. To in-

vestigate the catalytic mechanism and the efficiency

of an enzyme with changes in the parameters, and to

evaluate a suitable circumstance for assaying the

enzyme activity, the conditions should be kept con-

stant to allow reproducibility of data and to prevent a

misleading interpretation.

ENZYME, SUBSTRATE, ANDCOFACTOR

CONCENTRATIONS

In general, the substrate concentration should be

kept much higher than that of enzyme in order to

prevent substrate concentration dependency of the

reaction rate at low substrate concentrations. The

rate of an enzyme-catalyzed reaction will also show

linearity to the enzyme concentration when sub-

strate concentration is constant. The reaction rate

will not reveal the linearity until the substrate is

depleted, and the measurement in initial rate will be

invalid. Preliminary experiments must be performed

to determine the appropriate range of substrate con-

centrations over a number of enzyme concentrations

to prevent the phenomenon of substrate depletion. In

addition, the presence of inhibitor, activator, and

cofactor in the reaction mixture also influence

enzyme activity. The enzymatic activity will be low

or undetectable in the presence of inhibitors or in the

absence of activators or cofactors. In the case of

cofactors, the activated enzymes will be proportion-

al to the cofactor concentration added in the reaction

mixture if enzymes are in excess. The rate of reac-

Figure 7.6.The Eisenthal-Cornish-Bowden direct plot.

Figure 7.7.The Hill plot.