15.3 Analytical methods for the study of enzyme reactions
15.3.1 General considerations
Enzyme assays are undertaken for a variety of reasons, but the most common are:- to determine the amount (or concentration) of enzyme present in a particular
preparation (this is particularly important in diagnostic enzymology, Section 16.3); - to gain an insight into the kinetic characteristics of the reaction and hence to
determine a range of kinetic constants such asKm,Vmaxandkcat; - to study the effect of pH, temperature, inhibitors, etc. on the enzyme and to make
comparative studies of other enzymes that may be involved in a metabolic or
signalling pathway of which the enzymes are members. The study of enzyme
inhibition is fundamental to the development of new drugs.
Analytical methods for enzyme assays may be classified as eithercontinuous (kinetic)
ordiscontinuous (fixed-time). Continuous methods monitor some property change
(e.g. absorbance or fluorescence) in the reaction mixture, whereas discontinuous
methods require samples to be withdrawn from the reaction mixture and analysed
by some convenient technique. The inherent greater accuracy of continuous methods
commends them whenever they are available.
For simplicity, initial rates are sometimes determined experimentally on the basis of a
single measurement of the amount of substrate consumed or product produced in a given
time rather than by the tangent method. This approach is valid only over the short period
of time when the reaction is proceeding effectively at a constant rate. This linear rate
section comprises at the most the first 10% of the total possible change and clearly the
error is smaller the earlier the rate is measured. In such cases, the initial rate is propor-
tional either to the reciprocal of the time to produce a fixed change (fixed change assays)
or to the amount of substrate reacted in a given time (fixed time assays). The potential
problem with fixed-time assays is illustrated in Fig. 15.11, which represents the effect of
enzyme concentration on the progress of the reaction in the presence of a constant initial
substrate concentration (Fig. 15.11a). Measurement of the rate of the reaction at timet 0
(by the tangent method) to give the true initial rate or at two fixed times,t 1 andt 2 ,gives
the relationship between initial rate and enzyme concentration shown in Fig. 15.11b.
It can be seen that only the tangent method gives the correct linear relationship. Since the
correct determination of initial rate means that the observed changes in the concentration
of substrate or product are relatively small, it is inherently more accurate to measure
the increase in product concentration because the relative increase in its concentration is
significantly larger than the corresponding decrease in substrate concentration.15.3.2 Analytical methods for steady-state studies
Visible and ultraviolet spectrophotometric methods
Many substrates and products absorb light in the visible or ultraviolet region and the
change in the absorbance during the reaction can be used as the basis for the enzyme602 Enzymes