Example 1PRACTICAL ENZYME KINETICS
Question The enzymea-D-glucosidase isolated fromSaccharomyces cerevisiaewas studied
using the synthetic substratep-nitrophenyl-a-D-glucopyranoside (PNPG), which is
hydrolysed to releasep-nitrophenol which is yellow in alkaline solution (see Section
15.2.1 for further details). A 3 mM solution of PNPG was prepared and portions used
to study the effect of substrate concentration on initial rate using a fixed volume of
enzyme preparation. The total volume of each assay mixture was 10 cm^3 .A1cm^3
sample of the reaction mixture was withdrawn after 2 min, and placed in 4 cm^3
borate buffer pH 9.0 to stop the reaction and develop the yellow colour. The change
in absorbance at 400 nm was determined and used as a measure of the initial rate.
The following results were obtained:
PNPG (cm^3 ) 0.1 0.2 0.3 0.4 0.6 0.8 1.2
Initial rate 0.055 0.094 0.130 0.157 0.196 0.230 0.270What kinetic constants can be obtained from these data?AnswerSubject to the calculation of the molar concentration of PNPG in each reaction
mixture, it is possible to construct Lineweaver–Burk, Hanes and Eadie–Hofstee plots
to obtain the values ofKmandVmax. The fact that a 1 cm^3 sample of the reaction
mixture was used to measure the initial rate is not relevant to the calculation of [S].
Lineweaver–Burk, Hanes and Eadie–Hofstee plots derived from these data are shown
in Fig. 15.6 in whichv 0 measurements are expressed simply as the increase in
absorption at 400 nm. The plots giveKmvalues of approximately 0.2 mM andVmax
values of approximately 0.4.
As pointed out in Section 15.2.1,Vmaxvalues can be expressed in a variety of
units and their experimental value is dependent on a number of variables
particularly the concentration of enzyme. For comparative reasons,Vmaxis best
expressed in terms of the number of moles of product formed in unit time.
To do this, it is necessary to convert absorbance units to amount of product by
means of a Beer–Lambert law plot. Data for such a plot in this experiment are
given in Table A.Table A[PNP] (mM) 2.0 4.0 6.0 8.0 12.0 16.0 24.0
Absorbance (400 nm) 0.065 0.118 0.17 0.23 0.34 0.45 0.65A plot of these data confirms that the Beer–Lambert law is held and enables the
amount of product to be calculated. From this,v 0 values in units ofmmol min^1
can be calculated. The data for the three linear plots are presented in Table B.589 15.2 Enzyme steady-state kinetics