NAD+ -dependent Glyceraldehyde-3-phosphate + Dehydrogenase fromThermoproteus tenax.

1. Name the three enzymes that catalyze irreversible, regulatedreactions in glycolysis as studied in class.

2. What is the significance of the GAPDH reaction in E. coli toglycolysis?

3. How does the reaction catalyzed by GAPDH from T. tenaxpresented here differ from the reaction carried out in E. coli?

4. The activity of the GAPDH enzyme was assayed in the presenceof a constant amount of glyceraldehyde-3-phosphate and anincreasing 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. Additional data are given inTable 20.2.

a. Use the data in Figure 20.2 to estimate a KM value for theenzyme in the presence of these metabolites. Classify themetabolites listed in Table 20.2 as inhibitors or activators. Fillin your answers in the table provided. Explain how you decidedwhether these metabolites are inhibitors or activators, based onthe graph.

b. How would you classify NADH, ADP and ATP? (These data are notpresented in the graph). Are they inhibitors or activators? Addthis information to Table 20.2.

c. Explain the physiological significance of your answers toquestions 4a and 4b. NAD+ -dependent Glyceraldehyde-3-phosphate +Dehyd

5. In the absence of NADP+, the binding of NAD+ to the T.tenax GAPDH showed no cooperative binding (closed circles,Figure 2 above). In the presence of NADP however, the binding ofNAD+ to the T. tenax GAPDH was found to have a Hillcoefficient of 2 (open circles, Figure 2 above).

a) What is the significance of the change in the value of theHill coefficient?

b) Is this consistent with the shape of the curve and theinformation given in the background concerning the enzyme'squaternary structure?

6. What is the ATP yield for one mole of glucose oxidized by thepathway that uses the non-phosphorylating GAPDH enzyme?