2009_Book_FoodChemistry

140 2 Enzymes
Table 2.17. Enzyme concentrations used in the endpoint
method of enzymatic food analysis
Substrate Enzyme K m Enzyme
(mol/l)
concentration
(µcat/l)
Glucose Hexo- 1.0 · 10 −4 (30 ◦ C) 1.67
kinase
Glycerol Glycerol 5.0 · 10 −5 (25 ◦ C) 0.83
kinase
Uric acid Urate 1.7 · 10 −5 (20 ◦ C) 0.28
oxidase
Fumaric Fumarase 1.7 · 10 −6 (21 ◦ C) 0.03
acid
be prepared. In contrast to kinetic methods (see
below), the concentration of substrate which is
to be analyzed in food must not be lower than
the Michaelis constant of the enzyme catalyzing
the auxiliary reaction. The reaction time is
readily calculated when the reaction rate follows
first-order kinetics for the greater part of the
enzymatic reaction.
In a two-substrate reaction the enzyme is saturated
with the second substrate. Since Equation
2.41 is valid under these conditions,
the catalytic activity of the enzyme needed for
the assay can be determined for both one- and
two-substrate reactions. The examples shown in
Table 2.17 suggest that enzymes with low K m
values are desirable in order to handle the substrate
concentrations for the end-point method
with greater flexibility.
Data for K m and V are needed in order to calculate
the reaction time required. A prerequisite is
a reaction in which the equilibrium state is displaced
toward formation of product with a conversion
efficiency of 99%.
The determination of substrate using kinetic
methods is possible only as long as Equation 2.46
is valid. Hence, the following is required to
perform the assay:
a) For a two-substrate reaction, the concentration
of the second reactant must be so high
that the rate of reaction depends only on the
concentration of the substrate which is being
analyzed.
b) Enzymes with high Michaelis constants
are required; this enables relatively high
substrate concentrations to be determined.
c) If enzymes with high Michaelis constants are
not available, the apparent K m is increased by
using competitive inhibitors.
In order to explain requirement c), let us consider
the example of the determination of glycerol
as given in Table 2.16. This reaction allows
the determination of only low concentrations of
glycerol since the K m values for participating enzymes
are low: 6 ×10 −5 mol/l to3×10 −4 mol/l.
In the reaction sequence the enzyme pyruvate kinase
is competitively inhibited by ATP with respect
to ADP. The expression K m (1 +(I)/K I )
(cf. 2.5.2.2.1) may in these circumstances assume
a value of 6 ×10 −3 mol/l, for example. This corresponds
to an apparent increase by a factor of 20
for the K m of ADP (3 ×10 −4 mol/l). The ratio
(S)/K m (1 +[I]/K I ) therefore becomes 1 ×
10 −3 to 3×10 −2 . Under these conditions, the auxilary
reaction (Table 2.16) with pyruvate kinase
follows pseudo-first-order kinetics with respect to
ADP over a wide range of concentrations and, as
a result of the inhibition by ATP, it is also the ratedetermining
step of the overall reaction. It is then
possible to kinetically determine higher concentrations
of glycerol.
2.6.1.3 Kinetic Method
Substrate concentration is obtained using
a method based on kinetics by measuring the
reaction rate. To reduce the time required per
assay, the requirement for the quantitative conversion
of substrate is abandoned. Since kinetic
methods are less susceptible to interference than
the endpoint method, they are advantageous for
automated methods of enzymatic analysis.
2.6.2 Determination of Enzyme Activity
In the foreword of this chapter it was emphasized
that enzymes are suitable indicators for identifying
heat-treated food. However, the determination
of enzyme activity reaches far beyond this possibility:
it is being used to an increasing extent
for the evaluation of the quality of raw food and
for optimizing the parameters of particular food
processes. In addition, the activities of enzyme