N-acetylneuraminic acid aldolase of Clostridium perfringens: purification, properties and mechanism of action

A procedure for purifying N-acetylneuraminic acid aldolase of Clostridium perfringens is described. The purified enzyme has a molecular weight of 92,000 and consists of two protein components each of which has enzymatic activity. The purified aldolase has a pH optimum of 7.2 and a K_m of 1.75 mm; heavy metal ions are potent inhibitors. No metal ion requirement could be demonstrated for the enzyme and it is completely inhibited by sodium borohydride reduction in the presence of N-acetylneuraminic acid. This indicates that the enzyme is a Class I aldolase and forms a Schiff base-enzyme complex. There is no requirement for a substrate carboxyl group for binding to occur; substitution of a polar or bulky group at the deoxy position causes decreased binding. Substrate analog binding did not result in effective cleavage of the appropriate carbon-carbon bond. Kinetic studies with the pyruvate analog bromopyruvate are consistent with the existence of nucleophilic residues within the active site.