| E. coli is able to incorporate L-glyceraldehyde and L-glyceraldehyde 3-phosphate into phospholipids. L- 3-('3)H Glyceraldehyde was synthesized and the purity and the chemical identity of the product were checked by paper chromatography. L- 3-('3)H Glyceraldehyde 3-phosphate was synthesized from L- 3-('3)H glyceraldehyde in a reaction catalyzed by glycerokinase. E. coli extract contains a new enzyme activity which catalyzes an NADPH dependent reduction of L-glyceraldehyde 3-phosphate into sn-glycerol 3-phosphate. This enzyme, L-GAP reductase, was purified approximately 110 fold by ammonium sulfate precipitation followed by DEAE-Sephadex A-50 column chromatography. A procedure, specifically suitable for assaying the reductase activity in the crude extract, was developed. A more convenient spectrophotometric assay method was employed for the purified enzyme. The purified enzyme is free of NADPH dependent sn-glycerol 3-phosphate synthase, and contained only a trace of triosephosphate isomerase activity. Neither D-glyceraldehyde 3-phosphate nor dihydroxyacetone phosphate could act as a substrate. However when present in ten times the concentration of the normal substrate, L-glyceraldehyde 3-phosphate, both these compounds inhibited the enzyme activity by about 50%. The apparent K(,M) values for L-glyceraldehyde 3-phosphate and NADPH are 28 and 35 (mu)M respectively. A glyceraldehyde 3-phosphate analog, DL-3-hydroxy-4-oxobutyl-1-phosphonate was also found to be a substrate with an apparent K(,M) of 280 (mu)M. Three other carbonyl compounds, methylglyoxal, L-glyceraldehyde and D-glyceraldehyde are very poor substrates with apparent K(,M) values of 14, 28 and 100 mM respectively. At moderate concentrations sulfhydryl group inhibitors had no effect on the enzyme activity. At 100 (mu)M concentration Zn('+2) inhibited the enzyme activity by about 30% while Mn('+2) elevated the activity by about the same margin. Mg('+2), Ca('+2) and Fe('+2) were without effect at this concentration. L-Glyceraldehyde 3-phosphate is known to be bactericidal at 1.25 (mu)M concentration and the D-enantiomer is without effect. Furthermore, methylglyoxal is known to be bactericidal at or above 0.5 mM concentration. Strains of E. coli resistant to 1 mM methylglyoxal were isolated. The cell extract prepared from the mutant possessed increased capacity to transform methylglyoxal into D-lactate via a glutathione dependent reaction. These mutants were less sensitive to 2.5 mM DL-GAP suggesting that conversion of L-glyceraldehyde 3-phosphate into methylglyoxal may at least partly be responsible for the bactericidal acitivity of L-GAP. |
