Chem 454: Regulatory Mechanisms in Biochemistry Additional Problems – Chapter 24

1. The glyA – mutation in Chinese hamster ovary cells in tissue culture makes these cells partially dependent on . The mutation affects te mitochondrial form of transhydroxymethylase, which catalyzed the conversion of serine to glycine, with tetrahydrofolate serving as an acceptor of the hydroxymethyl group. Would you expect heme synthesis to be adversely affected in glyA – mutants? Explain?

2. Given an abundance of other amino acids in the diet, the a-keto acid analogs corresponding to the essential amino acids can substitute for the essential amino acids in the diet. a. Describe what makes and an essential amino acid. b. What do these observations tell yo about the steps in the synthesis of essential amino acids that may be missing in humans? c. If 15N-labeled alanine is supplied in the diet, many other amino acids in the body will contain at least a small amount of the label within 48 hours. What are primarily responsible for this observation?

3. In muscle, synthetase is very active, catalyzing the formation of glutamine from glutamate and ammonia at the expense of a molecule of ATP. In the liver, the rate of formation of glutamine is very low, but a high level of glutaminase activity, which generates ammonia and glutamate, is observed. How would you explain the different in the levels of activity in these two organs?

4. Consider three forms of bacterial : GS, the deadenylylated form; , GS–(AMP)1, a form with one AMP unit per 12 subunits; and GS–(AMP)12 the fully adenylated form. a. Which of these forms is most sensitive to feedback inhibition by several of the final products of glutamine metabolism, such as or ? Why is it important that the activity of the most sensitive form not completely inhibited by tryptophan? b. Why is it important that adenylyl transferase not carry out adenylylation and deadenylylation of glutamine synthetase at the same time? c. Glutamine synthetase in mammals is not subject to the same type of complex regulation that is seen in bacteria. Why?

5. Most of the synthesized in mammals contain all 20 common amino acids. More is degraded than is synthesized when even one essential amino acid is missing from the diet. a. Under such conditions, how could an increase in the rate of protein degradation provide the missing amino acid?

1 b. How does an increase in the rate of protein degradation contribute to increased levels of nitrogen excretion

6. The diagram below outlines the biosynthesis of a compound that is required for the oxidation of fatty acids in the mitochondrion:

CH2 CH2 CH2 NH3 H3C N CH3 H3C N CH3 a-ketoglutarate H3C N CH3 CH CH2 2 CH2 succinate + CO2 CH2 CH2 3B CH2 CH2 HO C H CH2 CH2 CH2 CH2 CH2 CH2 COO COO H C NH2 H C NH2 O COO COO 2

A C D

a. Name compound D and briefly explain its role in fatty acid metabolism? b. Name compound A. Why is it considered essential in human diets? c. Three molecules of compound B are required for the formation of compound C. Its synthesis depends on the availability of an essential amino acid. Name that amino acid and then name compound B and compound C.

7. A pathway for the synthesis of ornithine from glutamate is shown below:

O COO O H COO + + O H COO H3C C S CoA NADPH + H NADP H3N C H H3C C N C H H3C C N C H CH2 CH2 CH2 CH2 CH2 CH2 COO COO C O H Glutamate N-Acetylglutame N-Acetylglutamate g-semialdehyde

glutamate

a-ketoglutarate O H2O COO H3C C O O H COO

H3N C H H3C C N C H CH2 CH2 CH2 CH2 NH3 NH3

Ornithine N-Acetylornithine a. Why can this pathway also be considered to be part of the de novo pathway for the synthesis of ?

2 b. Inspect the pathway for biosynthesis given on page 674 of the text, and then explain why the N- of glutamate is needed for the synthesis of ornithine.

8. Elevated levels of ammonia in blood can result from deficiencies in one or another of the enzymes of the urea cycle. Measures taken to relieve hyperammonemia have included limiting intake of dietary proteins, administering a-keto analogs of several of the naturally occurring L-amino acids, or administering other compounds designed to exploit pathways of nitrogen metabolism and excretion a. In trying to determine why a patient has hyperammonemia, which organ should you check first for normal function? Why? b. Why would limiting protein intake assist in relieving chronic hyperammonemia? Why would eliminating dietary proteins altogether (without any other supplement) probably increase the level of hyperammonemia? c. Write a brief rationale for using a-keto acid analogs in treating hyperammonemia, mentioning a particular group of enzymes essential to your explanation. Would it be better to use a-keto analogs of essential or nonessential amino acids? Why?

9. Plants synthesize all 20 common amino acids de novo. Glyphospate, weed killer sold under the trade name Roundup, is an analog of phophoenolpyruvate that specifically inhibits 3-enolpyruvalyshikimate 5-phosphate synthase, a key enzyme of the pathway for chorismate biosynthesis. This compound is very effective plant herbicide, but has virtually no effect on mammals. Why?

10. A mutant of B. subtilis that lacks tryptophan synthetase can grow on minimal medium only when supplemented with exogenous tryptophan. Under these conditions, none of the intermediates in the tryptophan biosynthetic pathway from anthranilate to indole 3- glycerol phosphate are produced. However, when the bacteria have depleted the medium of tryptophan, the levels of those intermediates increase, even though there is no net production of tryptophan. Why?

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