JBC, Vol. 250, Issue 18, 7225-7230, Sep, 1975
On the role of substrate and GTP in the regulation of argininosuccinase activity
O. Rochovansky
As determined by equilibrium dialysis, bovine liver argininosuccinase of
molecular weight 202,000 binds 4 mol of argininosuccinate or arginine/mol
of enzyme. Negative homotropic interactions occur in the binding of both
ligands at 0.15 ionic strength in the presence of phosphate.
Argininosuccinate binds to two sites (Kdiss 1.6 times 10(-5) M) and four
sites (Kdiss 2.9 times 10(-4) M) at low and high substrate concentration.
Similarly, arginine binds to two sites (Kdiss 4.9 times 10(-4) M), and four
sites (Kdiss 1.6 times 10(-3) M). At 0.05 ionic strength in Tris-HCl
buffer, the four enzyme sites bind argininosuccinate independently and
arginine binding remains negatively cooperative. Kinetic analysis gave
double reciprocal plots that showed negative cooperatively also. The
changes in Km were analogous to changes in Kdiss, thus indicating that the
substrate binding sites correspond to catalytic sites. Since the
catalytically active enzyme is a tetramer composed of four identical or
closely similar subunits (Lusty, C.J., and Ratner, S. (1972) J. Biol. Chem.
247, 7010-7022), the present results show that each subunit contains one
catalytic site. Ionic strength, phosphate ions, and GTP have each been
found to influence negative cooperatively through a change in the affinity
for argininosuccinate. The significance of the negative homotropic
interactions and of the specific stimulation of activity by GTP is
discussed with respect to different conformational forms of the enzyme and
the in vivo regulation of argininosuccinase activity.
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