You discovered a novel enzyme URase that you suspect to be involved in sugar metabolism. You have already found in vitro assay conditions for accurate measurements of enzyme activity. However, you do not know its physiological substrate. Since the microorganism from which the enzyme was discovered thrives on fruits, it could be fructose. Or, it could be glucose. You will carry out kinetic experiments to determine which sugar molecule is the likely physiological the substrate of URase. Will you determine some kinetic parameters? What experiments will do you to determine those parameters? Provide experimental procedures without going into details, and how will you decide which sugar is the likely substrate? Explain your rationale.

You discovered a novel enzyme URase that you suspect to be involved in sugar metabolism. You have already found in vitro assay conditions for accurate measurements of enzyme activity. However, you do not know its physiological substrate. Since the microorganism from which the enzyme was discovered thrives on fruits, it could be fructose. Or, it could be glucose. You will carry out kinetic experiments to determine which sugar molecule is the likely physiological the substrate of URase. Will you determine some kinetic parameters? What experiments will do you to determine those parameters? Provide experimental procedures without going into details, and how will you decide which sugar is the likely substrate? Explain your rationale.

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter27: Metabolic Integration And Organ Specialization

Section: Chapter Questions

Problem 20P: Figure 27.3 illustrates the response of R (ATP-regenerating) and U (ATP-utilizing) enzymes to energy...

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A “pulse-chase” experiment using 14C-labeled carbon sources is carried out on a yeast extract maintained under strictly anaerobic conditions to produce ethanol. The experiment consists of incubating a small amount of14C-labeled substrate (the pulse) with the yeast extract just long enough for each intermediate in the fermentation pathway to become labeled. The label is then “chased” through the pathway by the addition of excess unlabeled glucose. The chase effectively prevents any further entry of labeled glucose into the pathway.(a) If [1-14C]glucose (glucose labeled at C-1 with 14C) is used as a substrate, what is the location of 14C in the product ethanol? Explain.(b) Where would 14C have to be located in the starting glucose to ensure that all the 14C activity is liberated as 14CO2 during fermentation to ethanol? Explain.
J. C. Servaites, in Plant Physiol. (1985) 78:839–843, observed that Rubisco from tobacco leaves collected before dawn had a much lower specific activity than the enzyme collected at noon. This difference persisted despite extensive dialysis, gel filtration, or heat treatment. However, precipitation of the predawn enzyme by 50% (NH4)2SO4 restored the specific activity to the level of the noon-collected enzyme. Suggest an explanation.
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