Food Chemistry

140 2 Enzymes

Table 2.17.Enzyme concentrations used in the end-
point method of enzymatic food analysis

Substrate Enzyme Km Enzyme

(mol/l) concen-

tration

(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
theMichaelisconstant 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 satu-
rated with the second substrate. Since Equa-
tion 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 Km
values are desirable in order to handle the sub-
strate concentrations for the end-point method
with greater flexibility.
Data for Kmand V are needed in order to calcu-
late the reaction time required. A prerequisite is
a reaction in which the equilibrium state is dis-
placed toward formation of product with a con-
version efficiency of 99%.

2.6.1.3 KineticMethod.........................................

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 con-
version 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.

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 concentra-

tion 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 highMichaelisconstants are

not available, the apparent Kmis increased by

using competitive inhibitors.

In order to explain requirement c), let us con-

sider the example of the determination of glyc-

erol as given in Table 2.16. This reaction allows

the determination of only low concentrations of

glycerol since the Kmvalues for participating en-

zymes are low: 6× 10 −^5 mol/lto3× 10 −^4 mol/l.

In the reaction sequence the enzyme pyruvate ki-

nase is competitively inhibited by ATP with re-

spect to ADP. The expression Km( 1 +(I)/KI)

(cf. 2.5.2.2.1) may in these circumstances assume

a value of 6× 10 −^3 mol/l, for example. This cor-

responds to an apparent increase by a factor of 20

for theKm of ADP (3× 10 −^4 mol/l). The ra-

tio(S)/Km( 1 +[I]/KI)therefore becomes 1×

10 −^3 to 3× 10 −^2. Under these conditions, the aux-

ilary 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 rate-

determining step of the overall reaction. It is then

possible to kinetically determine higher concen-

trations of glycerol.

2.6.2 DeterminationofEnzymeActivity.........................

In the foreword of this chapter it was emphasized

that enzymes are suitable indicators for identify-

ing heat-treated food. However, the determination

of enzyme activity reaches far beyond this pos-

sibility: 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