Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

4 Methods for Studying Enzymatic
Reactions
4.1 Introduction
Having established the general principles of enzyme structure and enzyme
catalysis, the remaining chapters will deal with each major class of enzymes
and their associated coenzymes, and a range of enzyme mechanisms will be
discussed. In this chapter we will meet the kind of experimental methods that
are used to study enzymes and to elucidate the mechanisms that will be given
later. There will be only a brief discussion of the biochemical techniques
involved in enzyme puriWcation and characterisation, since such methods are
described in much more detail in many biochemistry texts. The chapter will
focus on those experimental techniques that provide insight into the enzymatic
reaction and active site chemistry.
4.2 Enzyme purification
If we want to study a particular enzymatic reaction, the Wrst thing we need to do
is to Wnd a source of the enzyme and purify it. In order to test the activity of the
enzyme we must Wrst of all have an assay: a quantitative method for measuring
the conversion of substrate into product (Figure 4.1). In some cases conversion
of substrate to product can be monitored directly by ultraviolet (UV) spectroscopy,
if the substrate or product has a distinctive UV absorbance. Failing this, a
chromatographic method can be used to separate substrate from product and
hence monitor conversion. In order to quantify a chromatographic assay a
radioactive label is usually required in the substrate, so that after separation
from substrate the amount of product can be quantitated by scintillation
counting. Such an assay is highly speciWc and highly sensitive, but unfortunately
is rather tedious for kinetic work.
A more convenient assay for kinetic purposes is to monitor consumption of
a stoichiometric cofactor or cosubstrate, for example the cofactor nicotinamide
adenine dinucleotide (NADH) by UV absorption at 340 nm, or consumption of
oxygen by an oxygenase enzyme using an oxygen electrode. In other cases a
coupled assay is used, in which the product of the reaction is immediately
consumed by a second enzyme (or set of enzymes) which can be conveniently
monitored.
Once a reliable assay has been developed, it can be used to identify a
rich source of the enzyme, which might be a plant, an animal tissue, or
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