D. Nelson's exam  (70 pts)  Nov. 24, 1997                 name:                                            .


1.  (evolution I, 2 pts)
When comparing whole genome sequences, enzyme X was found only in the archaea.  
This enzyme is fundamental to all life.  Bacteria and eukarya both have this enzyme 
activity, but homologous sequences were not seen in the bacteria or the eukarya.  What can 
you say about this enzyme in the common ancestor to all life?

The enzyme was probably a ribozyme in the common ancestor.  It was later replaced by 
proteins that have the same activity but unrelated sequences, as we discussedd for 
ribonucleotide reductase.

2.  (amino acid biosynthesis II, 2 pts)
What is metabolic channeling?

Metabolic channeling is the migration of a substrate between multiple acitve sites in an 
enzyme complex, without release to the surrounding medium.  Example trp synthase alpha 
and beta subunits.

3.  (amino acid biosynthesis I, 2 pts)
A metabolic pathway for amino acid biosynthesis in Bacillus subtilis has three enzymatic 
steps.  By characterizing mutants in this pathway, you discover there are four 
complementation groups.  
	(a) What is a complementation group?
	(b) How do you explain your results?

Different auxotrophs (for the same substance like an amino acid, uracil, etc.) that cannot 
complement each other are said to be in the same complementation group.  This usually 
means in the same gene.  Four complementation groups in a pathway means four genes.  
Since there are three enzymes, one of them must be a dimer of non-identical subunits.

4.  (amino acid biosynthesis I, 4 pts)
In the regulation of glutamine synthase in E. coli, what is the role of the tetrameric protein 
PII?

PII is not an enzyme.  It can be uridylylated by an enzyme uridylyl transferase UT to 
become PIID that influences adenylyl transferase AT to deadenylylate GS and make it 
responsive to its feedback regulation system.  The uridylyl transferase is sensitive to the 
alpha ketoglutarate/glutamine ratio.  When glutamine is low the PII tetramer is converted to 
PIID.  When glutamine is high, PIID is deuridylylated to make PIIA.  PIIA switches AT to 
the adenylylation mode and it adds adenylyl groups to GS to inhibit it.  

PIIA also converts the kinase NRII to a phosphatase mode to remove phosphate from NRI-
P, a gene transcription factor needed to express GS.  So PIIA does two things.  It inhibits 
enzyme activity by promoting adenylylation of GS and it blocks gene expression of GS.

5.  (amino acid degradation and pyrimidine metabolism, 3pts)
There are two completely different uses for carbamoyl phosphate.  
	(a) What are they?  
	(b) What is the major difference in the reactions catalyzed by carbamoyl phosphate 
synthase I (CPS I) and CPS II? (I don't mean cellular location.)

It is used in pyrimidine biosynthesis and the urea cycle/arginine biosynthesis.
CPSI uses ammonia as a nitrogen source, while CPSII uses glutamine.

6.  (amino acid biosynthesis I and II, 2 pts)
What is a corepressor and how can it regulate amino acid biosynthesis?

A corepressor is a small molecule like an amino acid that binds to a gene regulatory protein, 
the aporepressor.  By binding it alters the protein structure so it will bind to a gene's 
promoter and repress its expression.  Many amino acids are corepressors of the committed 
step enzyme genes.  

7.  (amino acid degradation, 3 pts)
Amino groups are removed from amino acids in muscle, but it is toxic to transport 
ammonia in the blood.  Describe one way to deliver ammonia from muscle to the liver, so it 
can be eliminated by the urea cycle.

transaminate pyruvate to alanine.  Move to liver.  transaminate alanine to pyruvate making 
glutamate from alphaKG.  Use glutamate dehydrogenase to deaminate glutamate.

convert ammonia + glutamate to gluamine by glutamine synthase
move to liver and use glutaminase to remove the amino group as ammonia.

Some other creative answers were good for some credit.  

8.  (amino acid degradation, 3 pts)
Glycine is a two carbon amino acid, yet it is glucogenic, not ketogenic.  
	(a) What do glucogenic and ketogenic mean?  
	(b) Why is glycine degradation unusual?

Glucogenic amino acids are broken down to 3, 4 or 5 carbon compounds that can be made 
into glucose.  Ketogenic amino acids go to Acetyl CoA or acetoacetate and cannot be used 
to make glucose.  They form ketone bodies instead.  Glycine is unusual in that it is built up 
from a two carbon unit to a three carbon unit before it is broken down.  Thus it is 
glucogenic, even though it is a 2 carbon compound.

9.  (pyrimidine metabolism, 3 pts)
Describe the role of the active site sulfhydryl groups in ribonucleotide reductase.
How are these sulfhydryls restored to their original condition after formation of the 
deoxynucleotide?

The pair if SH groups in the active site are donors of hydrogen and electrons in the reaction 
mechanism.  They contribute to the replcement of the 2' OH with a hydrogen.  In the 
process they are oxidized to a disufide.  This can be restored by two different means.
1) Thioredoxin can reduce them and become oxidized itself.  This is reduced by thioredoxin 
reductase then this enzyme's disulfide is reduced by NADPH donating to an FAD.  
2) Glutaredoxin can reduce the disulfide in ribonucleotide reductase.  It is then restored by 
2 glutathione that become oxidized to GSSG.  The oxidized glutathione is reduced by 
glutathione reductase.

10.  (pyrimidine metabolism, 2 pts)
Why does 5-fluoro uracil block thymidylate synthase?

The compound is processed to 5 fluoro UMP.
F at the 5 position cannot leave during the reaction mechanism since it is so electronegative.  
The enzyme becomes trapped.  It is a suicide substrate.

11.  (pyrimidine metabolism, purine metabolism, 4 pts)
Methotrexate inhibits the purine de novo pathway and the formation of dTMP by 
thymidylate synthase.  
	(a) How does methotrexate block dTMP formation?
	(b) How does methotrexate block the purine de novo pathway?

It is a folate analog and it inhibits DHFR that is needed to convert DHF back to THF for 
thymidylate synthase.  Without N5 N10 methylene THF DNA synthesis is blocked.  
Purine synthesis requires N10 formyl THF, but it does not convert THF to DHF like 
thymidylate synthase.  Therefore, methotrexate does not affect purine synthesis directly.  
The reason the purine de novo pathway is inhibited is conversion of N10 formyl THF to 
N5 N10 methylene THF for thymidylate synthase.  This uses up the supply of N10 formyl 
THF and stops purine synthesis.

12.  (amino acid biosynthesis I, 3 pts)
Give three amino acids (and their TCA/glycolysis precursors) that are formed in one step 
by a transamination reaction.

pyruvate, alanine
oxaloacetate, aspartate
alpha KG, glutamate

13.  (pyrimidine metabolism, 3 pts)
In animals, CPS II is the committed step in pyrimidine biosynthesis.  In E. coli, aspartate 
transcarbamoylase is the committed step.  Why is there this regulatory difference between 
animals and E. coli?

E. coli has only one enzyme for both pathways.  It is at a branchpoint.  The next step is the 
best site to regulate these two pathways.  In animals, the CPSI and II are in different 
compartments and so they do not compete with each other.  CPSII is only used in 
pyrimidine synthesis, so it is a good site for regulation.

14.  (purine metabolism, 2 pts)
How does allopurinol relieve the symptoms of gout?

It is an hypoxanthine analog that blocks xanthine oxidase and reduces uric acid 
concentrations.  Precursors are more soluble and can be excreted.

15.  (purine metabolism, 3 pts)
What disease is caused by a defect in purine salvage?  Why does a loss of HGPRT activity 
cause the rate of purine synthesis to rise?

Lesch Nyhan syndrome (includes gout as part of the disease)
PRPP is not consumed by the salvage pathway causing PRPP to rise and activate the de 
novo pathway.

16.  (purine metabolism, 2 pts)
The first step in purine biosynthesis is formation of PRPP.  What kind of unusual mutation 
occurs in this enzyme (ribose-5-phosphate pyrophosphokinase) that leads to a human 
disease?

The enzyme is normally feedback inhibited by purine nucleotides.  The mutation blocks this 
inhibitory mechanism and leaves the enzyme constitutively on.  The disease is caused by 
superactivity rather than loss of activity.

17.  (amino acid biosynthesis II, 2 pts)
How does the Ames mutagenesis test work?

The histidine biosynthesis pathway is used to test for mutations.  Various mutants in the 
pathway are plated on his minus media and a substance is added.  The number of revertant 
colonies compared to a control shows the mutagenic potential of the compounds added.

18.  (amino acid biosynthesis II, 3 pts)
What are the two enzymes required for nitrate assimilation?  Where do they get their 
reducing equivalents?

nitrate reductase and nitrite reductase
In fungi and bacteria both enzymes get electrons from NADPH.  
In plants nitrate reducase uses NADH and nitrite reducase uses ferredoxin from 
photosynthesis.
Either answer is OK

19.  (amino acid biosynthesis II, 2 pts)
In E. coli, aromatic amino acid biosynthesis shares a key regulatory strategy with the 
pathway for lysine, methionine and threonine.  What is it?

Both have three different isoforms (DAHP and aspartokinase) that are regulated by the 
amino acid end products either by feedback inhibition or a corepressor mechanism

20.  (amino acid degradation II, 2 pts)
Aspartate acts as an amino group donor in the urea cycle and in the conversion of IMP to 
AMP.  Is this a transamination reaction?  What is the common product?

It is not a transamination since that would yield a keto acid.  This is a non-hydrolytic 
cleavage.  The common product is fumarate.

21.  (evolution I, 2 pts)
Why do carbon deposits with a 12C to 13C ratio higher than inorganic carbon sources 
indicate a biological origin for the carbon?

12C is preferentially incorporated into living beings by the process of carbon fixation.  This 
is an isotope effect.  There is no natural non-biological way to shift this ratio.

22.  (evolution I, 2 pts)
What is thought to have happened to the oxygen released by photosynthesis during the first 
2 billion years after photosystem II evolved?

It reacted with Fe2+ in the oceans forming banded iron formations and later on red beds.
This kept the atmospheric O2 concentration from rising until the Fe2+ was saturated.

23.  (evolution I, 2 pts)
What is the concept of the RNA world?

A world early in the history of life without protein or DNA, just RNA doing it all.

24.  (evolution I, 2 pts)
How can the idea of fusion between an archaebacterium and a eubacterium explain the 
origin of the eukaryotic nucleus?

If a gram negative bacterium without a cell wall phagocytosed an archaebacterium, the 
double membrane around the nucleus would be explained.  The archaebacterium would 
have to lose its cell wall and switch to ester based lipids from ether based lipids, and 
assume the genetic functions for the new cell.  The ER would also be a product of the 
invagination of the gram negative bacterium's plasma membrane.  

25.  (evolution II, 2 pts)
What evidence supports the idea that mitochondria and chloroplasts are bacteria that have 
become endosymbionts of eukaryotic cells?

rRNA sequences of mitochondria sort into the alpha proteobacteria on phylogenetic trees.
The rRNA of chloroplasts fall into the cyanobacteria in these trees.

These organelles also have a double membrane and heat shock proteins similar to bacteria.

26.  (evolution II, 3 pts) How have mutations in human mitochondrial DNA and the Y 
chromosome been used to infer the origins of modern humans?  What advantage is there to 
using this DNA? 

Mitochondrial DNA is inherited mother to daughter without recombination to confuse the 
effects of mutations that accumulate 17 times faster than nuclear DNA.  This makes it an 
ideal molecule to follow human evolution on a short time scale.  Trees made from mito 
DNA show the deepest branches to be from Africans, and estimates of mutation rate 
suggest 100,000 to 200,000 years as the time of origin of our mitochondrial DNA in a 
single woman.  

The Y chromosome has a similar part of its DNA only inherited from father to son.  
Mutations are much rarer here because it is nuclear DNA and also has no recombination.  
Studies of Y chromosome polymorphisms show that very few Africans have a rare 
mutation seen in apes but not in other humans, suggesting that this mutation changed in the 
human lineage soon after it branched from the apes.  This indicates an African origin.  
Trees made from the Y chromosome data also identify the 100,000 to 200,000 year time 
frame for the last common ancestor of our Y chromosome.  

27.  (evolution II, 3 pts)
How can genetic maps of different mammalian species be used to tell the evolutionary 
history of mammals?

The gene order can be compared (not the DNA sequence) and regions of same gene order 
called syntenic regions can be identified.  The breakpoints between these regions represent 
unique historical events in mammalian evolution and they can be used to follow the exact 
evolution of the mammals.  This will be more accurate than comparing sequences that have 
all diverged a similar degree and are not informative.

28.  (evolution II, 2 pts)
Researchers suspected that animals like centipedes achieved the large number of body 
segments by duplicating some of their homeobox genes.  This opinion has changed in the 
last year.  Why did it change, and what is the current hypothesis for these striking 
macroevolutionary changes?

Centipedes have the same nuber of HOM genes as insects, so the duplication of these genes 
is not responsible for the increase in segments.  Now the regulation of the HOM genes is 
thought to be responsible for these big changes in body segment number.

The following question was not given even as a bonus question because the test was 
already very long.

29.  Bonus question (not required, 2pts)  The amino acid biosynthesis pathways evolved 
before oxygen was in the atmosphere, and amino acids require no molecular oxygen in 
their synthesis.  The amino acid breakdown of the aromatic amino acids requires molecular 
oxygen.  These pathways must have evolved later, when oxygen was available in the 
atmosphere.  How do you think anaerobic organisms deal with excesses of aromatic amino 
acids?