2-Keto-3-deoxy-galactonate-6-phosphate Aldolase from Pseudomonas saccharophila

Abstract

This chapter describes an assay method and the purification procedure for 2-Keto-3-deoxy-galactonate-6-phosphate aldolase from Pseudomonas saccharophila. 2-Keto-3-deoxy-galactonate-6-phosphate (KDPGal) aldolase is found in extracts of galactose-grown Pseudomonas saccharophila along with 2-keto-3-deoxy-gluconate-6-phosphate (KDPG) aldolase. The two enzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose under conditions wherein the bulk of KDPGal aldolase activity is found in the column breakthrough, while much of the activity toward KDPG is retarded. Further separation is achieved by taking advantage of the instability of KDPG aldolase under mildly alkaline conditions. In contrast, KDPGal aldolase is completely stable under the conditions employed. Enzyme purification involves crude extract and heat step, DEAE-cellulose chromatography, dialysis, first chromatography on DEAE-sephadex, second chromatography on DEAE-sephadex, and chromatography on Sephacryl S-200. Catalysis by both KDPG and KDPGal aldolases of P. saccharophila are Schiff's-base mediated and their sole difference in overall stereomechanism is in the orientation of that carbonyl face of D-glyceraldehyde-3-phosphate presented to the pyruvyleneamine during the condensation–cleavage reaction. © 1982, Academic Press, Inc.