166 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
Most enzyme-catalyzed reactions are character-
ized by an increase in the rate of reaction and in-
creased thermal instability of the enzyme with
increasing temperature. Above the critical tempera-
ture, the activity of the enzyme will be reduced sig-
nificantly; while within this critical temperature
range, the enzyme activity will remain at a relatively
high level, and inactivation of the enzyme will not
occur. Since the rate of reaction increases due to the
increased temperature by lowering the activation
energy EA, the relationship can be expressed by the
Arrhenius equation: kAexp (EA/RT), where A
is a constant related to collision probability of reac-
tant molecules, R is the ideal gas constant (1.987
cal/mol—deg), T is the temperature in degrees
Kelvin (K °C 273.15), and krepresents the spe-
cific rate constant for any rate, that is, kcator Vmax.
The equation can be transformed into: lnKlnA
EA/RT, where the plot of lnKagainst 1/T usually
shows a linear relationship with a slope of EA/R,
where the unit of EAis cal/mol. The calculated EAof
a reaction at a particular temperature is useful in
predicting the activation energy of the reaction at
another temperature. And the plot is useful in judg-
ing if there is a change in the rate-limiting step of a
sequential reaction when the line of the plot reveals
bending of different slopes at certain temperatures
(Stauffer 1989).
Taken together, the reaction should be performed
under a constantly stable circumstance, with both
temperature and pH precisely controlled from the
start to the finish of the assay, for the enzyme to
exhibit highly specific activity at appropriate acid-
base conditions and buffer constitutions. Whenever
possible, the reactant molecules should be equili-
brated at the required assay condition following ad-
dition of the required components and efficient mix-
ing to provide a homogenous reaction mixture.
Because the enzyme to be added is usually stored at
low temperature, the reaction temperature will not
be significantly influenced when the enzyme volume
added is at as low as 1–5% of the total volume of the
reaction mixture. A lag phase will be noticed in
the rate measurement as a function of time when the
temperature of the added enzyme stock eventually
influences the reaction. Significant temperature
changes in the components stored in different envi-
ronments and atmospheres should be avoided when
the reaction mixture is mixed and the assay has
started.Figure 7.9.Plots of temperature effect on the energy
levels of reactant molecules involved in a reaction.
(A)The first plot depicts the distribution of energy levels
of the reactant molecules at room temperature without
the presence of an enzyme. (B)The second plot
depicts that at the temperature higher than room tem-
perature but in the absence of an enzyme. (C)The third
plot depicts that at room temperature in the presence of
an enzyme. The vertical line in each plot indicates the
required activation energy level for a reaction to occur.
The shaded portion of distribution in each plot indicates
the proportion of reactant molecules that have enough
energy levels to be involved in the reaction. The x-axis
represents the energy level of reactant molecules, while
the y-axis represents the frequency of reactant mole-
cules at an energy level.