Purification, Crystallization, and Properties of Adenylosuccinate Synthetase from Rat Skeletal Muscle

Abstract

Adenylosuccinate synthetase [EC 6.3.4.4] was purified and crystallized from rat skeletal muscle. The purified preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugal analysis, and by the latter procedure its average molecular weight was estimated to be 104,000 and its s₂₀,w value, 5.0. On SDS-polyacrylamide gel electrophoresis it gave only a single band with a molecular weight of 52,000. Sedimentation equilibrium experiments indicated that its molecular weight in 6 M guanidine-HCl was 49,000. Amino acid analyses indicated a subunit molecular weight of 52,860. These findings show that the native enzyme is composed of two subunits with molecular weights of 52,000. The optimum pH was 6.8 and the K_m values for L-aspartate, IMP, and GTP were calculated to be 2.5, 7.0, and 3.8 × 10⁻⁴ M, respectively. Nucleoside mono-and diphosphates, whether purine or pyrimidine derivatives, were potent inhibitors, while nucleoside triphosphates were not. The enzyme activity was inhibited considerably by succinate derivatives such as succinate, adenylosuccinate, and argininosuccinate. Succinate caused competitive inhibition with respect to aspartate with a K1 value of 4 × 10 M, and noncompetitive inhibition with respect to IMP and GTP with K1 values of 7.7 × 10⁻² M and 2.6 × 10⁻³ M, respectively. The purified enzyme preparation showed full activity in the presence of MgCl₂ and MgCl₂ could be partially replaced by MnCl₂ CoCl₂ or Cacl₂.