Using the coupled assay for pyruvate kinase activity, Christopher measured the t

Question

Using the coupled assay for pyruvate kinase activity, Christopher measured the total change in concentration of NADH for an assay to be 200 µM. How much PEP was consumed by pyruvate kinase (in µM to 3 s.f.)?

Steven was unsure whether his coupled assay for pyruvate kinase activity was working correctly. He added more lactate dehydrogenase to the cuvette and saw no change in the rate. Is this to be expected? yes or no

For the native lac operon, in the presence of high glucose and the unavailability of lactose:

Select one:

a. lacZ, lacY and lacA are transcribed

b. the lac repressor binds to the operator

c. RNA polymerase binds to the operator

d. lacI is not transcribed

Explanation / Answer

1. ) 1 mole of PEP produces one mole of pyruvate catalyzed by pyruvate kinase. Lactate dehydrogenase then converts One mole of pyruvate to one mole lactate simultaneously utilizing one mole of NADH. The reactions are working in 1:1 ratio. The change in concentration of NADH is 200 uM. It means 200 uM of NADH is being converted to NAD to convert equivalent amount i.e 200 uM of pyruvate to lactate.

200 uM of pyruvate will be produced by equivalent amount of PEP i.e 200 uM.

2. Yes. This a coupled reaction in which rate of pyruvate kinase activity is determined by measuring the reduction in NADH amount. Pyruvate kinase converts PEP to pyruvate. Lactate dehydrogenase then catalyzes the reduction of pyruvate to lactate by using electrons from NADH. The rate is ultimately dictated by the pyruvate kinase which provides the substrate for lactate dehydrogenase. If rate of pyruvate kinase activity is low, pyruvate will be produced slowly. Any increase in lactate dehydrogenase concentration would not have any effect as its substrate pyruvate is aready in low amount and most of enzyme will be in unbound state. Similarly if pyruvate kinase activity is high, pyruvate will be produced at a greater rate and most of the lactate dehydrogenase will be saturated. Therefore increase in lactate dehydrogenase amount will not have any effect on the rate.

3. In presence of high glucose and unavailability of lactose the lac repressor binds to the operator.

In presence of glucose cAMP levels will be low and cAMP receptor protein (CRP) will be inactivated as its allosteric regulator is in low aomunts. In active CRP will not bind to the promoter and is unable to recruit RNA polymerase to the lac promoter

Lactose is convert to allolactose by beta-galactosidase. Allolactose binds to lac repressor and prevents it from binding to the operator. In absence of lactose and hence allolactose, lac repressor will be in its active form and will bind to operator sequences in the lac operon.

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