Biology Reference
In-Depth Information
CHAPTER 4
ENZYME KINETICS
INTRODUCTION
Biochemists have isolated many enzymes from various biological sources
(Dixon and Webb, 1964), and molecular biologists have analyzed their
amino acid and nucleotide sequences together with their three-dimensional
structures, in order to understand their functions. Enzymes catalyze certain
chemical reactions, and have evolved over millions of years to carry out
their functions under required conditions very efficiently. The properties of
enzymes are thus specified by the rates of reactions they mediate. At the
same time, they may also promote reactions of similar chemicals but to a
less degree of efficiency.
For our present discussion, one of the glycerol dehydrogenases will be
considered (Fig. 4-1). It removes two hydrogen atoms from glycerol to
form dihydroxyacetone. The hydrogen atoms are delivered to a co-enzyme,
nicotine adenine dinucleotide (NAD). There are two nearby pockets on the
enzyme, one for glycerol and the other for NAD. The two small molecules
can bind to the enzyme sequentially. Then, they react and the products,
dihydroxyacetone and reduced NAD, will dissociate from the enzyme
molecule.
It is not easy to measure the concentration of dihydroxyacetone. Thus, the
rate of the enzymatic reaction is usually measured by the amount of reduced
NAD as a function of time. This molecule has a characteristic absorption of
light with wave-length at 340 nm. In a typical experiment, various amounts
of glycerol dehydrogenase, nicotine adenine dinucleotide and glycerol are
mixed in a buffer promoting this reaction at a pH not necessarily
physiological. The solution is then loaded into a UV-transparent glass
cuvette scanned by a spectrophotometer at 340 nm and recorded on a time
chart. The slope of the curve gives the velocity of the enzymatic reaction
for the given concentrations of the enzyme, co-enzyme and the substrate.
