Problem Set 2

1. Using structural formulae diagram how CO2 fixation occurs in the Calvin cycle.

A) If a microorganism is not a CO2 autotroph, what alternative mechanisms does it have to for CO2 fixation? Using chemical formulae, diagram this anapleurotic reaction in a cell which contains a pyruvate carboxlase and a cell which contains a PEP carboxylase.

B) Pyruvate and PEP caboxylases are activated by high concentrations of what molecule? Why are high concentrations of this molecule a good indicator of the cell's need for oxaloacetate?

C) For what anabolic and catabolic purposes do cells utilize oxaloacetate?

D) How do cells regulate whether the oxaloacetate will be used for catabolic or anabolic processes?

2. The second half of the glyoxalate cycle can be viewed as an anapleurotic reaction. What molecule is formed? Using structural formulae diagram these reactions.


3. Nitrogen metabolisms

A. What are the mechanisms of nitrogen assimilation using ammonia as the nitrogen source? What is the difference between the nitrogen atom in ammonia and the nitrogen atom in nitrate? What must occur to the nitrogen atom in nitrate before it can be it can be incorporated into the amino acids glutamate or glutamine?

B. Nitrogen assimilation is an extremely important process for microbial growth. Thus some microorganisms have evolved two interacting pathways, one that operates when ammonia concentrations are high and an alternate pathway that operates when ammonia concentrations are low. Discuss these two pathways and diagram these reactions using structural formulae. Name all enzymes.

C. The enzyme glutamine synthetase is a highly regulated enzyme. What two molecules influence the activity of these enzymes? How do these two molecules indicate the NH4+ concentration of the medium?

D. The proteins PII, uridylyl transfer/removal (UT/UR) enzyme and adenylyl transfer/removal (AT/AR) enzyme play a critical roles in the regulation of glutamine synthetase activity and transcription. Diagram these regulation pathways.

E. Starting with, a-ketoglutarate, ammonia and pyruvate.  Diagram, using structural formulae, the biosynthetic reactions needed to generate the amino acid alanine in a cell with and without the enzyme alanine dehydrogenase? Use coenzymes as needed.

4. Amino acid biosynthesis

A. The amino acid serine is made from the catabolic intermediate 3-phosphoglycerate, which is converted to the molecule 3-phosphohydroxypyruvate. Knowing the structure of pyruvate, one should be able to deduce the structure of 3-phosphohydroxypyruvate. Deduce this structure. After the formation of 3-phosphohydroxypyruvate, the synthesis of serine is mechanistically similar to the synthesis of aspartate. Using structural formulae diagram the amination reaction of 3-phosphohydroxypyruvate. How is this reaction similar to aspartate biosynthesis?

B. Sulfur must be in a reduced state before it is assimilated into organic compounds. However, environmental sources of sulfur are usually in oxidized states.  Once reduced sulfur is usually assimilated into the amino acid serine. Using structural formulae diagram that reaction?

C.  What amino acids require glutamic acid as an amine donor for their biosynthesis? What amino acids require glutamic acid as a direct intermediate in their synthesis?

E. Knowing the structure of glutamic acid, and how carbon atoms are designated in amino acids, one should be able to deduce the structure of glutamic gamma-semialdehyde. Glutamic gamma-semialdehyde is the intermediate in the synthesis of the amino acids proline. Using structural formulae, diagram this reaction.

F. Glutamic gamma-semialdehyde can also be converted to the amino acid ornithine. How does this reaction resemble a transamination reaction?

G. Although the amino acid homoserine is not found in proteins, it serves as an intermediate to many other amino acids. How is this amino acid related to aspartic acid? How must aspartic acid be modified to produce homoserine?

5. The regulation of amino acid biosynthesis.

A. How does feedback inhibition regulate the amino acid biosynthesis? What step in the biosynthetic pathway is usually the target of feedback inhibition? What thermodynamic property is usually associated with the targeted step?

B. Some enzymes, like glutamine synthetase, are regulated by a process called cumulative feedback inhibition. Describe how this process operates. What is unique about the products of enzymes that are regulated by cumulative feedback inhibition?


C. Tryptophan biosynthesis is regulated on several levels. The regulators of tryptophan biosynthesis are tryptophan and amino-acyl trypotophan tRNA. How does these molecules affect tryptophan biosynthesis?

D. Operons encoding catabolic enzymes and operons encoding anabolic enzymes are regulated differently. In general, which operons are under positive control and which operons are under negative control?

6. Aspartate transcarbamoylase.

A. Aspartate transcarbamoylase catalyses the committed step in pyrimidine biosynthesis, using structural formulae diagram the reaction. The product of the reaction then circularizes to form a 6-member ring, diagram that reaction.

B. The enzyme aspartate transcarbamoylase displays sigmoidal kinetics. How does this phenomenon relate to the structure of the enzyme and the number of active sites found in the complex and the affinity of the enzyme for its substrate?

C. If aspartic acid is abundant in the cell, what does this indicate about the about the state of protein synthesis? Why is the intracellular level of aspartic acid a good molecular indicator for protein synthesis?

D. If aspartic acid is at a low intracellular concentration, how will this affect the rate of pyrimidine biosynthesis?

E. Both ATP and CTP affect the activity of aspartate transcarbamoylase. What effects do each of these molecules have on the rate of enzyme activity? What is the evolutionary advantage of this regulation?