7 Enzyme Activities 165tion will not represent the total amount of enzyme
when the cofactor supplement is not sufficient, and
the situation can be avoided by adding excess co-
factors (Tipton 1992). Besides, loss of enzyme
activity may be seen before substrate depletion, as
when the enzyme concentration is too low and leads
to the dissociation of the dimeric or multimeric
enzyme. To test this possibility, addition of the same
amount of enzyme can be applied to the reaction
mixture to measure if there is the appearance of
another reaction progression curve.
EFFECTS OF PH
Enzymes will exhibit maximal activity within a nar-
row range of pH and vary over a relatively broad
range. For an assay of enzymatic catalysis, the pH of
the solution of the reaction mixture must be main-
tained in an optimal condition to avoid pH-induced
protein conformational changes, which lead to
diminishment or loss of enzyme activity. A buffered
solution, in which the pH is adjusted with a compo-
nent with pKaat or near the desired pH of the reac-
tion mixture, is a stable environment that provides
the enzyme with maximal catalytic efficiency. Thus,
the appropriate pH range of an enzyme must be
determined in advance when optimizing assay con-
ditions.
To determine if the enzymatic reaction is pH
dependent and to study the effects of group ioniza-
tions on enzyme kinetics, the rate as a function of
substrate concentration at different pH conditions
can be measured to simultaneously obtain the
effects of pH on the kinetic parameters. It is known
that the ionizations of groups are of importance,
either for the active sites of the enzyme involved in
the catalysis or for maintaining the active conforma-
tion of the enzyme. Plots of kcat, Km, and kcat/Kmval-
ues at varying pH ranges will reflect important infor-
mation about the roles of groups of the enzyme. The
effect of pH dependence on the value of kcatreveals
the steps of ionization of groups involved in the ES
complex and provides a pKavalue for the ES com-
plex state; the value of Kmshows the ionizing groups
that are essential to the substrate-binding step before
the reaction. Moreover, the kcat/Kmvalue reveals the
ionizing groups that are essential to both the
substrate-binding and the ES complex–forming
steps, and provides the pKavalue of the free reactant
molecules state (Brocklehurst and Dixon 1977,
Copeland 2000). From a plot of the kcat/Kmvalue on
the logarithmic scale as a function of pH, the pKa
value can be determined from the point of intersec-
tion of lines on the plot, especially for an enzymatic
reaction that is not associated with the Henderson-
Hasselbalch equation pH pKalog ([A-]/[HA])
(Fig. 7.8). Thus, the number of ionizing groups
involved in the reaction can be evaluated (Dixon and
Webb 1979, Tipton and Webb 1979).EFFECTS OFTEMPERATURETemperature is one of the important factors affecting
enzyme activity. For a reaction to occur at room tem-
perature without the presence of an enzyme, small
proportions of reactant molecules must have suffi-
cient energy levels to participate in the reaction (Fig.
7.9A). When the temperature is raised above room
temperature, more reactant molecules gain enough
energy to be involved in the reaction (Fig. 7.9B). The
activation energy is not changed, but the distribution
of energy-sufficient reactants is shifted to a higher
average energy level. When an enzyme is participat-
ing in the reaction, the activation energy is lowered
significantly, and the proportion of reactant mole-
cules at an energy level above the activation energy
is also greatly increased (Fig. 7.9C). That means the
reaction will proceed at a much higher rate.Figure 7.8.The effect of pH on the kcat/Kmvalue of an
enzymatic reaction that is not associated with the
Henderson-Hasselbalch equation.