The activity of the gapdh enzyme was assayed in the

Question

NAD⁺-dependent Glyceraldehyde-3-phosphate

Dehydrogenase from Thermoproteus tenax

1. a. The activity of the GAPDH enzyme was assayed in the presence of a constant amount of glyceraldehydes-3-phosphate and an increasing amount of NAD⁺. The activity of the control was compared to the activity in the presence of various metabolites. The results are shown in figure 20.2. Use the data in Figure 20.2 to estimate a K_M value of the enzyme in the presence of these metabolites. Classify the metabolites listed in the table below as inhibitors or activations. Explain how you decided whether these metabolites are inhibitors or activator based on the graph.

Metabolite

Apparent K_M  mM

Inhibitor or activator?

None

NADP⁺

Glucose-1-P

AMP

b. How would you describe the shape of the curve for NADP⁺. What might this imply?

Figure 20.2: Cosubstrate saturation of NAD⁺-dependent GAPDH of T. tenax in the presence of various effectors. Assay conditions were the following: 90 mM HEPES, pH = 7, 160 mM KCl, 4 mM DL-GAP (based on Brunner, et al., 1998).