Physical and Chemical Properties of β-Methylaspartase

A modified purification procedure for β-methylaspartase from Clostridium tetanomorphum, which gives electrophoretically pure enzyme in almost 50% yield, is described. 2. The z-average molecular weight of the native enzyme was determined to be 100,000 by the sedimentation equilibrium technique. The molecular weight was invariant in the pH range, 5.9 to 9.7, and was also not affected by ethylenediaminetetraacetate (10-4 m ) or by Mg++ and K+ at concentrations which are optimal for catalytic activity. 3. Under a variety of experimental conditions similar to those used in the sedimentation equilibrium studies, sedimentation velocity experiments yielded an s020,w value of 6.05 S for the native enzyme. 4. Trailing boundary sedimentation experiments showed that the sedimentation coefficient associated with β-methylaspartase activity was very close to 6.05 S. This result established that the enzyme species having an s020,w value of 6.05 S and a molecular weight of 100,000 was present, and catalytically active, under the conditions we have commonly used in steady state kinetic studies of the reaction. 5. The value of e279 was 5.63 x 104 m -1 cm-1 and the molar turnover number at pH 9.7, with Mg++ and K+ as activators, was 650 sec-1. 6. All catalytic activity was lost when 2 sulfhydryl groups, out of a total of 20 in the enzyme molecule, had reacted with p-chloromercuribenzoate.