FST 123
Problem Set 5
Spring, 2014
Name
Key
1. For each of the kinds of enzyme-catalyzed reaction listed below, indicate a particular cofactor that
commonly participates in the reaction. Fill in each blank.
a.acyl transfer reactions
Coenzyme A or Lipoic acid (Lipoamide)
b. methylation reactions
S-adenosyl methionine (maybe tetrahydrofolate)
c. transfer of one-carbon units, but not necessarily methyl groups.
tetrahydrofolate
d. two electron oxidation-reduction reactions
NAD+/NADH NADP+/NADPH FMN/FMNH2 FAD/FADH2 Vitamin C (ascorbic acid
(any three)
e. decarboxylation reactions
Pyridoxal phosphate Thiamine pyrophosphate (maybe biotin)
f. deamination reactions
Pyridoxal phosphate_ Just hydrolyze off the NH2 that was left on the PYP
g. one-electron transfer reactions
Flavin coenzymes, Coenzyme Q, heme, a few other metal ions
h. carboxylation reactions
Biotin
i. transamination reactions
Pyridoxal phosphate
2. Pyridoxal phosphate is involved in numerous reactions involving -amino acids. Using serine as an
example, the initial adduct between the amino acid and the cofactor might look like this:
beginning with this intermediate, "push electrons" to show how the following transformations
could be carried out.
a. decarboxylation
The scheme below is from an old doc that does not show the proton from the NH as being Hbonded to the phenolic oxygen (as show above). It is now believed that these groups remain
H-bonded throughout the arrow-pushing scheme. Remember, these are probably resonance
forms, anyway, not actual intermediates.
b. Elimination of -OH (from the CH2-OH of the serine moiety)
3. Bacteria in the genera Bacillus and Listeria have membranes whose phospholipids contain
branched-chain fatty acids in addition to the normal fatty acids. The synthesis of branched-chain
fatty acids begins with a branched-chain acyl CoA derived from one of the aliphatic amino
acids, valine, leucine or isoleucine. The initial step is the conversion of the amino acid into the
corresponding-ketoacid.
a. Show how valine could be converted to 2-ketoisovaleric acid (both shown below), and show
how the cofactor participates in the reaction (decide which cofactor the enzyme uses).
Valine
2-ketoisovalerate
-H
b. The next step involves oxidative decarboxylation of the 2-ketoisovalerate in the presence of
conezyme A to make the acyl CoA (shown below) that serves as the “primer” for the synthesis.
Indicate all cofactors that participate in the reaction, and show all of the steps that you can.
(Hint: the enzyme is analogous to the Pyruvate Dehydrogenase complex.)
As the hint says, this is just like Pyruvate dehydrogenase.
c. The acyl group is then transferred to Acyl Carrier Protein (ACP), which has a 4'phosphopantethiene at the active site. Then, two-carbon units from AcCoA are inserted,
and the fatty acid chain grows, with the branch remaining at the end of the fatty acid. If
you ignore the fact that the growing acyl chain is initially attached to CoA, and must be
transferred to ACP for each cycle, show how the stepwise insertion and subsequent
reactions occur that lead to a saturated branched-chain fatty acid (ester). Show each step
and each cofactor.
4. How could you determine if an enzyme contained a tightly bound flavin coenzyme?
Take an absorption spectrum and look for the peaks associated with one of the forms of a flavin.
5. Classify the following enzymes according to the Enzyme Commission nomenclature (i.e., give an
EC number).
a. alcohol dehydrogenase EC 1.1.1.1
b. trans aldolase EC2.2.1.2 (actually called a transferase, even though it involves
an addition/elimination-type mechanism
c. ribonuclease EC3.1.2.x
d. -amylase EC 3.2.1.2
e. phosphofructokinase EC 2.7.1.11
f. xylose isomerase EC 5.3.1.5
g. aminoacyl-tRNA synthetases (choose the valine enzyme). EC 6.1.1.9
6. This material is in the one of the last lectures, so you don't have to turn in the answer to this
problem. Consider it a practice question. Staphylococcal nuclease catalyzes the hydrolysis of
DNA to yield fragments terminating in a 3'-OH end and a 5'-phosphate end:
Way too easy … use 18O water and look to see if the heavy isotope shows up in the ribose or in the
phosphate. Mass spec would work. 31P-NMR would also work, but most people probably don’t
know that.