Question

NAD+ -dependent Glyceraldehyde-3-phosphate + Dehydrogenase from Thermoproteus tenax.

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

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

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

4. The activity of the GAPDH enzyme was assayed in the presence of a constant amount of glyceraldehyde-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. Additional data are given in Table 20.2. a. Use the data in Figure 20.2 to estimate a KM value for the enzyme in the presence of these metabolites. Classify the metabolites listed in Table 20.2 as inhibitors or activators. Fill in your answers in the table provided. Explain how you decided whether these metabolites are inhibitors or activators, based on the graph. b. How would you classify NADH, ADP and ATP? (These data are not presented in the graph). Are they inhibitors or activators? Add this information to Table 20.2. c. Explain the physiological significance of your answers to questions 4a and 4b. NAD+ -dependent Glyceraldehyde-3-phosphate + Dehyd

5. The Hill coefficients for NAD binding + to the T. tenax GAPDH in the presence and absence of NADP were measured + and are shown on the graph in Figure 20.2.

What is the significance of the change in the value of the Hill coefficient? Is this consistent with (a) the shape of the curve and (b) the information given in the background on the enzyme?s structure?

6. What is the ATP yield for one mole of glucose oxidized by the pathway that uses the nonphosphorylating GAPDH enzyme?

Answer 1

Q1) The three enzymes that catalyze irreversible, regulated reactions in glycolysis are

i) Hexokinase

ii) Phosphofructokinase

iii) Pyruvate kinase

the reactions catalysed by these enzymes are

Q2) In E.coli during glycolysis, the enzyme Glyceraldehyde-3-phosphate dehydrogenase reversably converts the substrate Glyceraldehyde-3-Phosphate into 1,3-BisPhosphoglycerate as shown below:

Q3) In Thermoproteus tenax, the enzyme GAPDH catalyses the irreversible conversion of Glyceraldehyde-3-phopsphate to 3 phosphoglycerate in a phosphate-independent manner

In E.coli, the reaction catalyzed is a reversible and phosphate dependent reaction. In Thermoproteus tenax, it is an irrevesible phosphate independent reaction.

Q4 and Q5 - data tables and graphs not provided.

Q6. the glycolytic pathway in which the non-phosphorylating GAPDH enzyme is used will have no net gain of ATP molecules because initially the two ATP molecules will be consumed and at the end only two ATP will be produced resulting in net gain of zero ATP molecules. However, NADH will be produced.