Purification and Characterization of Pyridoxal Kinase from Vitamin B_6 Auxotroph of Escherichia coli

Abstract

Pyridoxal kinase was purified from Escherichia coli KG 980 by ammonium sulfate fractionation, and successive chromatographies on DEAE-cellulose, Sephadex G-150 and pyridoxamine (PM)-Sepharose. The overall purification was about 2,500-fold over the crude extract with an yield of about 23%. Polyacrylamide gel electrophoresis indicated that the final product was 70 to 75% homogeneous. The molecular weight of the enzyme was estimated to be 47,000 by Sephadex G-100 gel filtration and 58,000 by SDS polyacrylamide gel electrophoresis. From isoelectric gel electrophoresis, an isoelectric point of 4.8 was estimated. The K_m values for pyridoxine (PN), pyridoxal (PL) and PM were 9.1 × 10^<-6>M, 1.3 × l0^<-4>M and 4.2 × 10^<-4>M, respectively. The relative V_<max> values obtained for PN, PL and PM were 100%, 70% and 170%, respectively. The pH optimum was 6.0 with PN or PL and 7.0 with PM as the substrate. PM, 4-deoxypyridoxine (dPN) and 5'-dPN showed competitive inhibition for phosphorylation of PN with Ki values of 4.4 × l0^<-4>M, 2.5 × l0^<-5>M and 2.9 × l0^<-4>M, respectively. Pyridoxal 5'-phosphate showed noncompetitive inhibition with Ki Value of 9.1 × 10^<-4>M. PL and 5'-deoxypyridoxal (dPL) were both competi-tive and noncompetitive inhibitors for PN. The K_i Values of PL was 1.4 × 10^<-4>M (as a competitive inhibitor) and 5.9 × l0^<-5>M (as a noncompetitive inhibitor), and the K_i Values of 5'-dPL was 2.5 × l0^<-4>M (as a competitive inhibitor) and 3.8 × 10^<-5>M (as a noncompetitive inhibitor).