section 7.2
Allosteric Enzyme Regulation
113
1.
Aspartate transcarbamoylase
(ATCase) is an
allosteric enzyme of the bacterium
Escherichia coli,
which has been extensively studied. This enzyme
catalyzes the transfer of the carbamoyl group from
carbamoyl phosphate to the a-amino group of
aspartate:
o
o
H
I
l
I
I
+1
H2N—C—O—P—0 “ + H3N—C—COO'
I
I
0 '
CH2
I
C00"
Carbamoyl phosphate
Aspartate
0
I
HO—P—0 '
I
O '
Inorganic phosphate (P,)
C02 +
Glutamine
+
ATP
Carbamoyl phosphate
Aspartate—-^
N- Carbamoyl aspartate
;
i
t
Uridine monophosphate (UMP)
Uridine triphosphate (UTP)
o
T
J
Cytidine triphosphate (CTP)
FIGURE 7-4
Regulation of pyrimidine nucleotide biosynthesis in
E. coli.
0
H
11
1
+
H2N— C— N— C— COO" + H +
H
I
CH2
I
COO'
N-Carbamoyl aspartate
This committed reaction, catalyzed by a regulatory
enzyme, is the second step (Figure 7-4) in the
biosynthesis of pyrimidines. N-carbamoylaspartate
ultimately is converted to the pyrimidine nucleotide
cytidine triphosphate (CTP), which is the negative
modulator of the enzyme. That is, as the CTP
concentration increases, it inhibits the ATCase by
decreasing its affinity for substrates. The preceding is
an example of an allosteric feedback inhibition.
ATCase has a molecular weight of 310,000 and
consists of 12 polypeptide chains (subunits). The
catalytic and the regulatory sites reside on two
different sets of subunits: six larger subunits
(M.W. 33,000) have catalytic sites, and six smaller
subunits (M.W. 17,000) have regulatory sites. X-ray
crystallographic studies of ATCase by Lipscomb
showed that the molecule is roughly triangular. The
six catalytic subunits (C) are present as two trimers
(C
3
), one above the other (but not exactly parallel),
with the dimeric regulatory subunits (R
2
) forming an
equatorial belt around the edges of the trimers
(Figure 7-5). In the center of the molecule, an
aqueous cavity is accessible through several openings.
ATCase can be dissociated into two unequal clusters
with different subunit arrangements by treatment with
mercurials (e.g., p-hydroxymercuribenzoate). The
larger cluster containing three catalytic subunits (C
3
)
is catalytically active. The v versus [S] plot exhibits
a hyperbolic profile and is insensitive to CTP. The
smaller cluster is a dimer of regulatory subunits (R
2
)
and, as expected, shows no catalytic activity but does
bind to CTP. The native enzyme can be reconstituted
with return of the allosteric activity by removal of the
mercurial compound and mixing of both catalytic and
regulatory clusters:
2C
3
+
3R
2
->
R
6
C
6
Catalytic
Regulatory
Native
cluster
cluster
enzyme
Reconstitution also requires zinc ions (Zn2+), since
the native enzyme contains six atoms of Zn2+. C
3
and
R
2
clusters can be dissociated further into their
respective, inactive monomeric subunits by strong
denaturing agents (Figure 7-5).
ATCase can assume different conformations
depending on whether it is active or inactive. In the
presence of the substrate or positive modulator, it is
in a catalytically more active conformation known as
the
relaxed state
or
R-state.
In the presence of the
negative modulator, the enzyme is in less active
conformation known as the
taut state
or
T-state.
The allosteric kinetic effects of ATCase are
shown in Figure 7-6. The interaction of substrates
with the enzyme is cooperative (an example of
homotropic cooperativity), as indicated by the
sigmoidal shapes of the v versus [S] plots, CTP
being an inhibitor and ATP an activator. These
modulators compete for the same regulatory site
and modulate the affinity of the enzyme for its
