Exam Key for Dr. Nelsons second exam Nov. 19, 1998

1) The first step in de novo purine biosynthesis makes PRPP, but it is not the committed 
step in this pathway.

a. Why is this first step not the committed step?
	PRPP is used in several other pathways so it cannot be the committed step.

b. One mutant in this enzyme is superactive.  What does that mean?
	The enzyme is not feedback inhibited so it is constitutively on.

c. Why does the superactive enzyme cause gout?
	Overproduction of PRPP causes activation of purine biosynthesis.  The breakdown 
product of these nucleotides is uric acid which causes gout by precipitation in joints.

2) The purine nucleotide cycle converts IMP to AMP in two steps of the de novo pathway,
then it reforms IMP by a deamination step.

a. What is the purpose of the purine nucleotide cycle?
	It replenishes TCA cycle intermediates especially in muscle.

b. What intermediate is generated in this cycle?
	fumarate

3a) Why are there two carbamoyl phosphate synthase enzymes in eukaryotes?
	CPSI is used in the urea cycle and CPSII is used for pyrimidine biosynthesis.

3b) Where are they in the cell?
	CPSI is in mitochondria. CPSII is in the cytosol.

3c) What are the sources of nitrogen used by the two enzymes?
	CPSI uses ammonia. CPSII uses glutamine

4) Ribonucleotide reductase has a selectivity regulatory site and an activity regulatory site.

a. What two nucleotides compete at the activity regulatory site?
	ATP and dATP

b. When dGTP is bound at the selectivity regulatory site, what is the specificity of the 
active site?
	ADP is converted to dADP (ATP was also accepted, though it is not technically 
correct)

5) Most genetic diseases caused by defects in amino acid metabolism are caused by defects 
in amino acid degradation, not amino acid biosynthesis.  Why is that?
	Missing amino acids can be obtained in the diet, but breakdown products cannot be 
eliminated if a breakdown pathway is blocked.  The intermediates may be toxic, or they 
may be neurotransmitters that have harmful effects at high concentrations.

6) In tryptophan synthase, glycerol phosphate is removed from an indole ring on the alpha 
subunit and replaced by serine on the beta subunit to make tryptophan.  If radioactive 
indole is added to the enzyme reaction, no radioactivity is ever found in the final product.  
What process is responsible for this?
	Metabolic channeling

7) What is the purpose of the urea cycle?
	To convert toxic ammonia to less toxic urea for nitrogen elimination.  Also, it is part 
of Arginine biosynthesis.

8) What happens in a transamination reaction?
	An amino group is transfered to a keto acid to form a new amino acid and a 
different keto acid, as in glutamate + oxaloacetate going to alpha ketoglutarate and 
aspartate.  Pyridoxal phosphate is the coenzyme in these reactions.

9) What evidence suggests that hydrogenosomes are derived from mitochondria?
	The heat shock proteins Hsp10, Hsp60 and Hsp70 from hydrogenosomes are most 
like mitochondrial heat shock proteins on phylogenetic trees.  This indicates they had a 
common ancestor.

10) Archaea from hot environments contain ether linked lipids, while bacteria and eukarya 
have ester linked lipids.

a. What is one possible explanation for the ether linked lipids?
	These are optimal for maintaining the correct membrane fluidity and integrity at high 
temperatures.

b. Archaea have been found by PCR of sea water samples that are not from a hot 
environment.  Do you think they will have ether linked lipids?
Both yes and no answers were acceptable if you argued for them.
	Yes, the evolution of ester linkages was probably a very rare event and it is not 
likely to happen easily, so the cooler environment archaea will probably still have ether 
linked lipids.  They may use other means to adjust fluidity.  

	No, the cooler environment will drive the evolution of ester linked lipids to maintain 
lipid fluidity.

11) DNA chip technology and nylon filter microarrays are powerful methods, but they are 
not the answer to every question.  Give two examples where they would fail to detect 
important changes.
	Posttranslational regulation of proteins would not be detectable by checking mRNA 
levels.  Phosphorylation, or feedback inhibition would not show up in mRNA changes.
Subtle changes of 2 fold or less would also not be detectable.

12) What is the incentive for spending millions of dollars to engineer a complex pathway 
into yeast when a good chemical/bioconversion process already exists to make cortisol?
	The current method makes cortisol in 8 steps.  Saving even one step can make a 
more efficient cost savings to a company.  To save all 8 steps and go from galactose to 
cortisol would be a spectacular savings.  Its worth lots of money.