Data Interpretation

The following data were obtained in studies on the effects of pH on the enzyme
Glucose-6-phosphate dehydrogenase.

Studies were carried out at pH 7.6 and pH 9.0 and the reaction followed by measuring
the increase in absorbance of light at 340nm due to NADPH production.

Substrate concentration Increase in Absorbance in the first 5min

X 104 (M)
pH 7.6 pH 9.0
0.174 0.062 0.029
0.267 0.075 0.04
0.526 0.089 0.075
1.666 0.11 0.13
4.000 0.12 0.17

Questions
1. Construct a properly labeled lineweaver-Burk plot from the data.

Fig 1) Shows the two velocities of glucose-6-phosphate dehydrogenase at both pH 7.6

& 9 at an Abs of 340nm and a substrate conc. of 1x10^4M. These figures show the
increase of the first 5 minutes and reciprocals were then taken to plot a linear graph.
 2. Calculate the Km and Vmax values for both pH’s and comment on the effect of
pH on this enzyme.

pH 7.6

Vmax = 1/(1/V) = 1/8.2369 = 0.12150

Km = Km/V x Vmax =1.3834 x 0.12150 = 0.16795

pH 9.0
Vmax = 1/(1/V) = 1/4.3225 = 0.23134
Km = Km/V x Vmax =5.2907 x 0.23134 = 1.22399

The optimum pH of glucose-6-phosphate dehydrogenase is 7.4 and this due to this

being one of the enzymes used in the physiological glycolic pathway of glucose to
pyruvate.

Looking at normal bell-curve graph that relates the enzyme velocity related to pH, it
is noticed that pH 7 gives a better affinity and as the pH decreases or increases, the
affinity of the enzyme falls dramatically. The pH 7.6 in the experiment is closer to the
biological pH environment that it would usually reside in, providing a higher Km
value than that of pH9 which is too alkaline for the normal functionality of the
enzyme due to a disruption of the hydrogen ions becoming polarized and essentially
denaturing the enzyme.

pH 7.6 gives a lower Vmax value than that of pH 9 as the affinity of the enzyme is
greater in relation to the substrate as the conditions that the enzyme are present in are
optimal. pH 9 has a higher Vmax value and this is due to the fact that the enzyme has
a much harder time binding to the substrate mainly due to the deformation of the
structural binding sites which would require an increase in substrate resulting in a
lower affinity.