section 27.6
Regulation of Purine Biosynthesis
625
Regeneration of the ribonucleotide reductase is ac-
complished in
E scherichia co li
and in mammals by
thioredoxin, a dithiol polypeptide (M.W. 12,000) coen-
zyme, which also plays a role in other protein disul-
fide reductase reactions. In thioredoxin, two cysteine
residues in the sequence -Cys-Gly-Pro-Cys are con-
verted to cystine. Reduced thioredoxin is regenerated by
thioredoxin reductase, a flavoprotein enzyme that uses
NADPH + H+.
E. coli
mutants unable to synthesize thioredoxin are
still able to form deoxyribonucleotides. In these bacte-
ria, a related substance, glutaredoxin, and two molecules
of glutathione carry out the reduction. In
L actobacillus,
the triphosphate is reduced and vitamin Bi
2
is an es-
sential coenzyme. Another example of this use of vita-
min B
] 2
is in
Euglena,
where the diphosphates are re-
duced. The mammalian system is nearly identical to that of
E. coli.
Ribonucleotide reductase from
E. co li
consists of two
subunits, Bi and B2, neither of which possesses catalytic
function. Bj is a dimer in which each monomer contains a
substrate binding site and two types of allosteric effector
binding sites. One type of effector site confers substrate
specificity and the other is regulatory. B] contains a pair of
sulfhydryl groups that are required for catalytic activity.
B
2
also is a dimer, contains one nonheme Fe(III), and has
an organic free- radical delocalized over the aromatic ring
of a tyrosine residue in each of its polypeptide chains. The
catalytic site is formed from the interaction of Bi and B2; a
free-radical mechanism involves the tyrosyl residues, iron
atom of B2, and sulfhydryl groups of B i. An antineoplastic
agent,
hydroxyurea,
inhibits ribonucleotide reductase by
inactivating the free radical.
Ribonucleotide reductase is regulated so as to en-
sure a balanced supply of deoxynucleotides for DNA
synthesis. For example, if excess dATP is present, de-
creased synthesis of all the deoxyribonucleotides en-
sues, whereas ATP stimulates formation of dCDP and
dUDP. Binding of TTP (also designated as dTTP) stim-
ulates formation of dGDP and hence of dGTP; binding
of dGTP stimulates the formation of dADP and hence of
dATP. In this way, these nucleotide effectors, by bind-
ing to various regulatory sites, tend to equalize the con-
centrations of the four deoxyribonucleotides required for
DNA synthesis.
27.6 Regulation of Purine Biosynthesis
Regulation of
de novo
purine biosynthesis is essential be-
cause it consumes a large amount of energy as well as of
glycine, glutamine, N10-formyl FH4, and aspartate. Regu-
lation occurs at the PRPP synthetase reaction, the ami-
dophosphoribosyltransferase reaction, and the steps in-
volved in the formation of AMP and GMP from IMP.
PRPP Synthetase Reaction
PRPP synthetase requires inorganic phosphate as an al-
losteric activator. Its activity depends on intracellular con-
centrations of several end products of pathways in which
PRPP is substrate. These end products are purine and
pyrimidine nucleotides (Figure 27-12).
Increased levels of intracellular PRPP enhance
d e novo
purine biosynthesis. For example, in patients with HPRT
deficiency, the fibroblasts show accelerated rates of
purine formation. Several mutations of PRPP synthetase,
which exhibit increased catalytic activity with increased
production of PRPP, have been described in gouty subjects.
Amidophosphoribosyltransferase Reaction
This reaction is the first and uniquely committed reac-
tion of the
d e novo
pathway and the rate-determining
step. Amidophosphoribosyltransferase is an allosteric en-
zyme and has an absolute requirement for a divalent
cation. The enzyme is inhibited by AMP and GMP, which
bind at different sites. The enzyme also is inhibited by
pyrimidine nucleotides at relatively high concentrations.
R ib o se 5 -p h o sp h a te + ATP
.
P R P P + G lutam ine
5 -P h o sp h o rib o sy la m in e
i
|
(M ultiple ste p s)
1
.IM P.
_
x ® —
-
A M P -S ©
XM P
.1
1
'
-A M P
I
\
1
\
G M P =
V©
I
l
- A D P
I
l
\
G D P -
\
I
I
- A T P
l
G T P - -
FIG URE 27-12
Feedback regulation of the
de novo
pathway of purine biosynthesis. Solid
lines represent metabolic pathways, and broken lines represent sites of
feedback regulation. © , Stimulatory effect; © , inhibitory effect. Regu-
latory enzymes: A, PRPP synthetase; B, amidophosphoribosyltransferase;
C, adenylosuccinate synthetase; D, IMP dehydrogenase.
