Food Biochemistry and Food Processing (2 edition)

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7 Biocatalysis, Enzyme Engineering and Biotechnology 133

Figure 7.6.A schematic diagram showing the free energy profile of the course of an enzyme catalysed reaction involving the formation of

enzyme–substrate (ES) and enzyme–product (EP) complexes. The catalysed reaction pathway goes through the transition states TS 1 ,TS 2 ,

and TS 3 , with standard free energy of activationGc, whereas the uncatalysed reaction goes through the transition state TSuwith standard

free energy of activationGu.

constants at a given temperature and a given enzyme concentra-

tion. TheKmor Michaelis constant is the substrate concentration

at whichv=Vmax/2 and its usual unit is M. TheKmprovides

us with information about the substratebinding affinityof the

enzyme. A highKmindicates a low affinity and vice versa (Moss

1988, Price and Stevens 1999).

TheVmaxis the maximum rate of the enzyme-catalysed re-

action and it is observed at very high substrate concentrations

where all the enzyme molecules are saturated with substrate, in

the form of ES complex. Therefore

Vmax=kcat[Et]

where [Et] is the total enzyme concentration andkcatis the rate

of breakdown of the ES complex (k+ 2 in the equation), which

is known as theturnover number.kcatrepresents the maximum

number of substrate molecules that the enzyme can convert to

product in a set time. TheKmdepends on the particular en-

zyme and substrate being used and on the temperature, pH,

ionic strength, etc. However, note thatKmis independent of the

enzyme concentration, whereasVmaxis proportional to enzyme

concentration. A plot of the initial rate (v) against initial substrate

concentration ([S]) for a reaction obeying the Michaelis–Menten

kinetics has the form of a rectangular hyperbola through the ori-

gin with asymptotesv=Vmaxand [S]=−Km(Fig. 7.9A). The

termhyperbolic kineticsis also sometimes used to characterise

such kinetics.

There are several available methods for determining the pa-

rameters from the Michaelis–Menten equation. A better method

for determining the values ofVmaxandKmwas formulated by

Hans Lineweaver and Dean Burk and is termed the Lineweaver-

Burk (LB) ordouble reciprocal plot(Fig. 7.9B). Specifically, it

is a plot of 1/vversus 1/[S], according to the equation:

1

v

=

Km

Vmax

·

1

[S]

+

1

Vmax

Such a plot yields a straight line with a slope ofKm/Vmax.

The intercept on the 1/vaxis is 1/Vmaxand the intercept on the

1/[S]axisis− 1 /Km.

The rate of an enzymatic reaction is also affected by changes

in pH and temperature (Fig. 7.10). When pH is varied, the veloc-

ity of reaction in the presence of a constant amount of enzyme is

typically greatest over a relatively narrow range of pH. Since en-

zymes are proteins, they possess a large number of ionic groups,

which are capable of existing in different ionic forms (Labrou

et al. 2004a). The existence of a fairly narrow pH-optimum for