Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.