BLBS102-c08 BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come
8 Enzyme Activities 171Figure 8.2. (A)Plot of progress curve during an enzyme-catalyzed
reaction for product formation.(B)Plot of progress curves during an
enzyme-catalyzed reaction for product formation with a different
starting concentration of substrate.(C)Plot of reaction rate as a
function of substrate concentration measured from slopes of lines
from(B).establishment of the steady state of a reaction. When the reac-
tion is approaching equilibrium or the substrate is depleted, the
rate of product formation decreases, and the slope strays away
from linearity. As is known by varying the conditions of the
reaction, the kinetics of a reaction will appear linear during a
time period in the early phase of the reaction, and the reaction
rate is measured during this phase. When the substrate concen-
trations are varied, the product formation profiles will display
linear substrate dependency at this phase (Fig. 8.2B). The ratesfor each substrate concentration are measured as the slope of a
plot of product formation over time. A plot of initial rate as a
function of substrate concentration is demonstrated as shown in
Figure 8.2C. Three distinct portions of the plot will be noticed: an
initial linear relationship showing first-order kinetic at low sub-
strate concentrations; an intermediate portion showing curved
linearity dependent on substrate concentration, and a final por-
tion revealing no substrate concentration dependency, i.e., zero-
order kinetic, at high substrate concentrations. The interpretation
of the phenomenon can be described by the following scheme:
The initial rate will be proportional to the ES complex concentra-
tion if the total enzyme concentration is constant and the substrate
concentration varies. The concentration of the ES complex will
be proportional to the substrate concentration at low substrate
concentrations, and the initial rate will show a linear relationship
and substrate concentration dependency. All of the enzyme will
be in the ES complex form when substrate concentration is high,
and the rate will depend on the rate of ES transformation into
enzyme-product and the subsequent release of product. Adding
more substrate will not influence the rate, so the relationship of
rate versus substrate concentration will approach zero (Brown
1902).However, a lag phase in the progress of the reaction will be
noticed in coupled assays due to slow or delayed response of the
detection machinery (see below). Thoughvcan be determined
at any constant concentration of substrate, it is recommended
that the value of substrate concentration approaching saturation
(high substrate concentration) be used so that it can approach its
limiting valueVmaxwith greater sensitivity and prevent errors
occurring at lower substrate concentrations.
Measurement of the rate of enzyme reaction as a function of
substrate concentration can provide information about the ki-
netic parameters that characterize the reaction. Two parameters
are important for most enzymes—Km, an approximate measure-
ment of the tightness of binding of the substrate to the enzyme;
andVmax, the theoretical maximum velocity of the enzyme reac-
tion. To calculate these parameters, it is necessary to measure the
enzyme reaction rates at different concentrations of substrate, us-
ing a variety of methods, and analyze the resulting data. For the
study of single-substrate kinetics, one saturating concentration
of substrate in combination with varying substrate concentra-
tions can be used to investigate the initial rate (v), the catalytic
constant (kcat), and the specific constant (kcat/Km). For inves-
tigation of multisubstrate kinetics, however, the dependence of
von the concentration of each substrate has to be determined,
one after another; that is, by measuring the initial rate at vary-
ing concentrations of one substrate with fixed concentrations of
other substrates.
A large number of enzyme-catalyzed reactions can be ex-
plained by the Michaelis-Menten equation:v=Vmax[S]
(Km+[S])=kcat[E][S]
(Km+[S]),where [E] and [S] are the concentrations of enzyme and sub-
strate, respectively (Michaelis and Menten 1913). The equation
describes the rapid equilibrium that is established between the
reactant molecules (E and S) and the ES complex, followed by