130 2 Enzymes
In order to better understand the form of the
enzyme involved in catalysis, a hypothetical
enzyme-substrate system will be assayed and
interpreted. We will start from the assumption
that data are available for v 0 (initial velocity)
as a function of substrate concentration at
several pH’s, e. g., for the Lineweaver and
Burk.ThevaluesforKmand V are obtained
from the family of straight lines (Fig. 2.32) and
plotted against pH. The diagram of K−m^1 =f(pH)
depicted in Fig. 2.33a corresponds to Fig. 2.31c
which implies that neutral (En) and positively
charged (En+^1 ) enzyme forms are active in
binding the substrate.
Figure 2.33b: V is dependent on one prototropic
group, the pK value of which is below neutral-
ity. Therefore, of the two enzyme-substrate com-
plexes, En+^1 AandEnA, present in the equilib-
rium state, only the latter complex is involved in
the conversion of A to the product.
In the example given above, the overall effect of
pH on enzyme catalysis can be illustrated as fol-
lows:
(2.79)This schematic presentation is also in agreement
with the diagram of V/Km=f(pH)(Fig. 2.33c)
Fig. 2.32.Determination of V and Kmat different pH
values
Fig. 2.33.Evaluation of Kmand V versus pH for a hy-
pothetical casewhich reveals that, overall, two prototropic
groups are involved in the enzymecatalyzed
reaction.
An accurate determination of the pK values of
prototropic groups involved in enzyme-catalyzed
reactions is possible using other assays (cf.J.R.
Whitaker, 1972). However, identification of these
groups solely on the basis of pK values is not
possible since the pK value is often strongly in-
fluenced by surrounding groups. Pertinent to this
claim is our recollection that the pH of acetic acid
in water is 4.75, whereas in 80% acetone it is
about 7. Therefore, the enzyme activity data as
related to pH have to be considered only as pre-
liminary data which must be supported and veri-
fied by supplementary investigations.2.5.4 InfluenceofTemperature.................................
Thermal processes are important factors in the
processing and storage of food because they al-
low the control of chemical, enzymatic and mi-
crobial changes. Undesired changes can be de-
layed or stopped by refrigerated storage. Heat
treatment may either accelerate desirable chem-
ical or enzymatic reactions or inhibit undesirable
changes by inactivation of enzymes or microor-
ganisms. Table 2.12 informs about quality dete-