Essential Cell Biology 5th edition

144
HOW WE KNOW
MEASURING ENZYME PERFORMANCE
At first glance, it seems that a cell’s metabolic pathways
have been pretty well mapped out, with each reaction
proceeding predictably to the next. So why would
anyone need to know exactly how tightly a particular
enzyme clutches its substrate or whether it can process
100 or 1000 substrate molecules every second?
In reality, metabolic maps merely suggest which pathways
a cell might follow as it converts nutrients into
small molecules, chemical energy, and the larger building
blocks of life. Like a road map, they do not predict
the density of traffic under a particular set of conditions;
that is, which pathways the cell will use when it is starving,
when it is well fed, when oxygen is scarce, when it
is stressed, or when it decides to divide. The study of an
enzyme’s kinetics—how fast it operates, how it handles
its substrate, how its activity is controlled—allows us to
predict how an individual catalyst will perform, and how
it will interact with other enzymes in a network. Such
knowledge leads to a deeper understanding of cell biology,
and it opens the door to learning how to harness
enzymes to perform desired reactions, including the
large-scale production of specific chemicals.
Speed
The first step to understanding how an enzyme performs
involves determining the maximal velocity, V max , for the
reaction it catalyzes. This is accomplished by measuring,
in a test tube, how rapidly the reaction proceeds in
the presence of a fixed amount of enzyme and different
concentrations of substrate (Figure 4–36A): the rate
should increase as the amount of substrate rises until
the reaction reaches its V max (Figure 4–36B). The velocity
of the reaction can be measured by monitoring either
how quickly the substrate is consumed or how rapidly
the product accumulates. In many cases, the appearance
of product or the disappearance of substrate can be
observed directly with a spectrophotometer. This instrument
detects the presence of molecules that absorb light
at a particular wavelength; NADH, for example, absorbs
light at 340 nm, while its oxidized counterpart, NAD + ,
does not. So, a reaction that generates NADH (by reducing
NAD + ) can be monitored by following the formation
of NADH at 340 nm in a spectrophotometer.
Looking at the plot in Figure 4–36B, however, it is difficult
to determine the exact value of V max , as it is not
clear where the reaction rate will reach its plateau. To
get around this problem, the data are converted to their
reciprocals and graphed in a “double-reciprocal plot,”
where the inverse of the velocity (1/v) appears on the
y axis and the inverse of the substrate concentration
(1/[S]) on the x axis (Figure 4–36C). This graph yields
a straight line whose y intercept (the point where the
line crosses the y axis) represents 1/V max and whose x
intercept corresponds to –1/K M . These values are then
converted to values for V max and K M .
Control
Substrates are not the only molecules that can influence
how well or how quickly an enzyme works. In
many cases, products, substrate lookalikes, inhibitors,
and other small molecules can also increase or decrease
(A)
(B)
(C)
increasing [S]
v = initial rate of
substrate consumption
(µmole/min)
v =
V max [S]
K M + [S]
–1/K M
1/v (min/µmole)
1/V max
K M
1/v = (1/[S]) +1/V max
V max
[S] (µM)
1/[S] (µM –1 )
Figure 4–36 Measured reaction rates are plotted to determine the V max and K M of an enzyme-catalyzed reaction. (A) Test
tubes containing a series of increasing substrate concentrations are prepared, a fixed amount of enzyme is added, and initial reaction
rates (velocities) are determined. (B) The initial velocities (v) plotted against the substrate concentrations [S] give a curve described
by the general equation y = ax/(b + x). Substituting our kinetic terms, the equation becomes v = V max [S]/(K M + [S]), where V max is the
asymptote of the curve (the value of y at an infinite value of x), and K M is equal to the substrate concentration where v is one-half V max .
This is called the Michaelis–Menten equation, named for the biochemists who provided evidence for this enzymatic relationship. (C) In
a double-reciprocal plot, 1/v is plotted against 1/[S]. The equation describing this straight line is 1/v = (K M /V max )(1/[S]) + 1/V max . When
1/[S] = 0, the y intercept (1/v) is 1/V max . When 1/v = 0, the x intercept (1/[S]) is –1/K M . Plotting the data this way allows V max and K M to
be calculated more precisely. By convention, lowercase letters are used for variables (hence v for velocity) and uppercase letters are
used for constants (hence V max ).
ECB5 04.36