Adenylosuccinate synthetase (
IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4) was purified about 750-fold to a homogeneous state from
Yoshida sarcoma ascites tumor cells. A yield of 38% purified
enzyme was achieved by a procedure including affinity chromatography on
hadacidin-
Sepharose 4B. Ultracentrifugal analyses showed that the molecular weight of the native
enzyme was 102 000 with an s20,w value of 4.5 and that the molecular weight in 6 M
guanidine-HCl was 47 000. These values indicate that the native
enzyme is composed of two subunits. The isoelectric point was determined to be 5.9 by isoelectric focusing. The optimum pH for activity was 6.8-7.0. The Km values for
IMP,
aspartate and
GTP were calculated to be 4.1, 9.8 and 0.7 . 10(-4) M, respectively. The
antibiotic,
hadacidin was strongly inhibitory, causing competitive inhibition with respect to
aspartate with a Ki value of 2.5 . 10(-6) M.
Nucleoside mono- and
diphosphate also inhibited the
enzyme activity, but their inhibitions were not apparently specific. The purified
enzyme showed full activity in the presence of Mg2+, and Mg2+ could be partially replaced by Mn2+, Co2+, Ca2+ or Cu2+. Divalent
metal ions, such as Cd2+, Pb2+, Zn2+, Cu2+ and Mn2+, interfered with the activity by antagonizing Mg2+. Hg2+ or
PCMB inactivated the
enzyme, suggesting that an SH-group may be important for activity.