Purification and Properties of Neurospora Adenylosuccinase

A procedure is described for the isolation of Neurospora adenylosuccinase. Under the conditions employed in the isolation of this protein, it exists in at least three forms that can be easily separated by ultracentrifugation or by moving boundary electrophoresis. The 10S form and the 4.0S form are readily interconvertible by changes in the hydrogen ion concentration. Only one form of the enzyme appears to catalyze the adenylosuccinate → AMP + fumarate reaction, and the same form catalyzes the S-AICAR → AICAR + fumarate reaction. (Here, the symbol S denotes a succinyl group and AICAR denotes 5-amino-4-imidazolecarboxamide ribonucleotide.) The active form of the enzyme has a molecular weight of about 200,000, optimal activity at pH 8.3, and a 0° stability optimum at pH 8.0. The Km for adenylosuccinate is 1.2 x 10-6 m. The inactive forms of the enzyme have molecular weights of about 27,000 and 55,000 and appear to be subunits of the 200,000 molecular weight form on the basis of identical peptide maps, amino acid composition, and the fact that the relative amounts of the three peaks in the ultracentrifuge can be modified by changing the pH. The results of the amino acid composition studies and the peptide maps of trypsin digests of adenylosuccinase indicate that the smallest molecular form of the enzyme is 25,000 to 30,000 molecular weight. This molecular weight value was confirmed by sedimentation equilibrium analyses, which gave a value of 27,600. On the basis of these data, the active form of adenylosuccinase consists of 6 to 8 identical subunits.