David Nelson's exam over Amino Acid Metabolism/ Molecular Evolution
5 lectures, 50 points Nov. 25, 1996

1) [1 pt] In nitrogen fixation by nitrogenase, eight electrons are consumed to make two
ammonium ions from molecular nitrogen N2. The oxidation state of N2 is zero and the
oxidation state of NH4+ is -3. This means that only six electrons are needed. What
happens to the other two electrons?

They are used to make molecular hydrogen from two protons. The hydrogen must leave
the active site before N2 can bind.

2) There are two different types of feedback inhibitors for GS.
[1 pt] What are the two types? Six compounds made from glutamine and three amino
acids that are not made from glutamine.
[1 pt] Why do these two classes function as feedback inhibitors?
One set is a product glutamine and should indicate glutamine levels and therefore
the need for more glutamine. The other set are indicators of nitrogen abundance
in the cell and they should indicate the general need for glutamine to be used in
synthesis.
[1 pt] Why doesn't a monomer of GS have enzymatic activity?
The glutamine synthase active site is at the interface of two identical subunits.

3) [3 pts] Given the following hypothetical structures, convert the delta carboxyl into an
amine. [Hint this should take three steps (only two intermediates) that you have seen
frequently in amino acid metabolism.] Tell what type of reaction is occuring at each of the
three steps, such as decarboxylation, oxidation, etc.

Step 1. activate the COO- group by phosphorylation (or some other method of activation)
Step 2. Reduce the activated carboxyl to an aldehyde and release the PO4. (reduction)
Step 3. Transaminate the aldehyde to make the amine. (transamination)

4) [1 pt] In the synthesis of ornithine from glutamate, the first reaction is an acetylation of
the alpha amino group. Why is it necessary to do this?

The alpha amino group can react with the aldehyde created by reduction of the carboxyl
position (just as we did in question 3). Therefore it must be blocked before the aldehyde is
made. Proline biosynthesis does not block the alpha amino group and it cyclizes with the
terminal aldehyde.

5) [2 pts] In the urea cycle, there are two nitrogens that contribute to the urea molecule.
What is the source of the urea nitrogens? [arginine is not an acceptable answer]

An ammonia nitrogen comes from the carbamoyl phosphate. The other nitrogen is from
aspartate that reacts with citrulline to form argininosuccinate.

6) [2 pts] What are the two ways to remove amino nitrogen from amino acids.

1. transamination
2. oxidative deamination

7) [2 pts] Which of the following compounds is not made from chorismate? [may be more
than one]
A. lignin
B. plastoquinone
C. tryptophan
D. vitamin C not made from chorismate
E. prephenate
F. anthranilate
G. shikimate not made from chorismate (chorismate is made from shikimate)

8) [2 pts] In E. coli there are three DAHP synthase genes and each is feedback inhibited by
one of the three aromatic amino acids. In Bacillus subtilis, there is only one DAHP
synthase. On the diagram, show how the aromatic amino acid pathway is feedback
inhibited in B. subtilis.

In your notes I described a simplified regulation of this pathway. Prephenate and
chorismate feedback inhibit DAHP synthase, and prephenate dehydratase is inhbited by
phe, while prephentate dehydrogenase is inhibited by tyr.

That is what I am looking for here.

There is a little more complexity to the regulation given in your text.

Prephenate and chorismate also feedback inhibit shikimate kinase and prephenate also
inhibits chorismate mutase.

I was not expecting this extra information on the exam.

9) [3pts] Draw out the three carbamoyl phosphate synthase I reactions.
See your text p. 843 for structures.

Bicarbonate + ATP ----> carbonyl phosphate + ADP

Carbonyl phosphate + NH3 -----> Carbamate + P04

Carbamate + ATP -----> Carbamoyl phosphate + ADP

10) [3 pts] For each metabolic disease list the amino acid pathway that is affected. Please
include if it is a biosynthetic pathway or a breakdown pathway.

alkaptonuria tyrosine breakdown

porphyria heme biosynthesis

albinism tyrosine breakdown (phenylalanine also acceptable)

11) [2 pts] Name two pieces of evidence that suggest the tryptophan biosynthetic enzymes
are expressed as a single protein complex.

1) non-sequential enzymes in the pathway are covalently linked in some species
2) different sequential enzymes or subunits are fused together in different species.
3) some subunits have no activity unless present in a dimer with other subunits.
(anthranilate synthase alpha and beta or tryptophan synthase alpha and beta)
4) channeling of substrates supports the idea of a complex.

12) [2 pts] Describe briefly how glutathione-S-transferase is used in a popular protein
purification method.

Your favorite gene is over-expressed in E. coli as a fusion protein with GST. A matrix
with glutatione is used to purify GST from the cell lysate, bringing your favorite protein
along with it. Proteolytic digestion with factor Xa or thrombin releases your protein from
GST at a specific engineered site at the fusion joint.

13) [3 pts] Describe how aspartokinase is regulated at the level of gene expression [not
feedback inhibition].

The three aspartokinase genes are co-repressed by different products of the diverging
pathway for lysine, isoleucine, methionine and threonine biosynthesis. Thr and Ile co-
repress aspartokinase I, Met co-represses aspartokinase II and lysine co-represses
aspartokinase III.

14) [1 pt] All the alpha amino groups in amino acid biosynthesis come from glutamate.
What is one way that glutamate can be synthesized?

1) glutmate dehydrogenase can couple ammonia to alpha ketoglutarate using NADPH
2) glutamate synthase can transfer glutamine's amide group to alpha ketoglutarate to make
two glutamates (one from alpha ketoglutarate and one from glutamine). This also requires
NADPH.

15) [2 pts] Name four relics of the RNA world

1) ATP, 2) ribosomal RNA, 3) The RNAse P RNA subunit, 4) The signal recognition
particle RNA, 5) tRNAs, 6) messenger RNAs, 7) nucleotide containing coenzymes, 8)
splicosomal RNAs, 9) Telomerase RNA, 10) self splicing introns.

16) [1 pt] In the last common ancestor to all life, the ribonucleotide reductase enzyme is
thought to be an RNA ribozyme. Why?

All three domains of life have these enzymes, but they are not related in sequence,
suggesting they were replacing an RNA ribozyme after divergence of the three domains.

17) [2 pts] The tree of life has three main branches. To find the root of any tree requires an
outgroup. What did molecular evolutionists do to root the tree of life.

Ancient genes that had duplicated before the last common ancestor were used to root the
tree. These included alpha and beta subunits of the F1FO ATPase, and the proteins EF-Tu
and EF-G, elongation factors in protein synthesis. These genes served as an outgroup to
the duplicated gene's tree: Ef-Tu sequences were an outgroup to EF-G sequences and vice
versa.

18) [2 pts] According to Gupta and Golding's model for eukaryote evolution, what is the
source of the nucleus and what is the source of the cytoplasm?

The nucleus is proposed to be from an Archaebacterium and the cytoplasm from a gram
negative bacterium.

19) [3 pts] Why does Christian de Duve propose in his book Vital Dust that bacteria and
archaea split before 3.5 billion years ago? [hint photosynthesis is important]

Stromatolites that are 3.5 billion years old have been identified. These stromatolites look
like modern day stromatolites that contain cyanobacteria. The inference is that
cyanobacteria were present 3.5 billion years ago in these fossil stromatolites.
Cyanobacteria are photosynthetic. So photosynthesis predates 3.5 billion years.
Chlorophyll is not found in Archaea and in few bacteria, so the Archaea and bacteria are
assumed to have separated before photsynthesis evolved. Therefore, they split before 3.5
billion yeasrs ago. The 3.75 billion year old banded iron deposits also suggest PSII
photosynthesis was operating at this ancient time. So the split between Archaea and
bacteria may be before 3.75 billion years ago.

20) [1 pt] What is the significance of the banded iron formations seen in sediments dated
from 3.75 to 1.7 billion years ago?

The banded iron formations are thought to be formed by oxygen released by
photosynthesis reacting with ferrous iron in the ancient oceans. These deposits are then an
indicator of when water splitting (PSII) photosynthesis evolved.

21) [1 pt] What evidence suggests that hydrogenosomes had a common ancestor with
mitochondria?

The Hsp70, Hsp60 and Hsp10 sequences from hydrogenosomes match best with the
sequences of these proteins from mitochondria. Therefore, both organelles are thought to
have a common ancestor among the alpha subdivision of the proteobacteria.

22) [2 pts] The introns early model was promoted by Walter Gilbert. He was very pleased
that an intron had been found in a mosquito gene for triose phosphate isomerase where he
predicted an intron would be found. How have more recent results from additional insects
discredited the introns early model?

Finding an intron in this position in an introns early model suggested that the same intron
should be present in many other insect TIM genes. When this was looked for, it was seen
only in the closest relative to the Culex mosquito, Aedes, but not in more distant relatives
like Anopheles, moths and flies. This argues for a recent insertion of this intron in the
ancestor to Culex and Aedes mosquitoes, not an ancient origin.

23) [3 pts] The mouse small eye gene can substitute for the Drosophila eyeless gene and
cause normal development of a fly eye in a Drosphila eyeless mutant. How can the mouse
small eye gene be a homolog of the Drosophila eyeless gene, when flies and vertebrates
diverged 600 million years ago, before eyes existed?

The eyeless gene predates the formation of eyes in any species. Its function is to start a
developmental program in a light sensitive tissue. Therefore, it is a master switch not a
gene that codes for eyes directly. The ancestor of flies and vertebrates had a light sensitive
tissue that was controlled by this gene, and it still controls the development of this tissue,
even though the eyes that form are very different. Eyes are thought to have evolved
independently about 40 times in different lineages.

24) [3 pts] Mitochondrial Eve has been misunderstood by many to mean there was a single
woman (or a very small population of humans) about 200,000 years ago in Africa and she
is the mother of us all. Why is this interpretation incorrect?

This interpretation confuses gene lineages with individual lineages. There was a single
woman whose mitochondrial DNA was the direct ancestor of all our mitochondrial DNA.
However, she was not the ancestor of all our 70,000 to 100,000 nuclear genes. These
have been contributed by thousands of individuals. The population size necessary to
maintain the highly polymorphic DRB1 alleles (59 different alleles) seen in humans today is
estimated at 100,000 individuals over the course of human evolution.