Food Biochemistry and Food Processing (2 edition)

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BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come


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
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