Glyceraldehyde phosphates are important intermediates of many central metabolic pathways in a large number of living organisms. One key intermediate during glycolysis is D-glyceraldehyde 3-phosphate (D-GAP), a three-carbon sugar phosphate-ester that can as well be found in a variety of other pathways. The opposite enantiomer, L-glyceraldehyde 3-phosphate (L-GAP), has been shown to be a substrate of YghZ. This enzyme catalyzes the stereospecific and NADPH-dependent reduction of L-GAP in an alternative pathway to the formation of dihydroxyacetone phosphate and the detoxification of L-GAP, which inhibits enzymes in phospholipid biosynthesis and glycolysis in E. coli.1-3
In contrast to D-GAP, no chemical synthesis of enantiomerically pure L-GAP is available until now. However, it was shown that L-glyceraldehyde (L-GA) can be phosphorylated in an ATP/PEP coupled enzymatic reaction with glycerokinase.4 The disadvantage of the existing methods is that no L-GAP has been isolated and that it has to be freshly prepared each time.
Here, we investigated the substrate specificity of glycerokinase from Cellulomonas sp.by quantitative31P-NMR spectroscopy and found a nearly 100% conversion of L-GA to L-GAP. Racemic DL-glyceraldehyde did not provide comparable results due to the ability of D-glyceraldehyde (D-GA) to catalyze the dephosphorylation of phosphoenolpyruvate (PEP) in the presence of the enzyme.5 By using pure D-GA as substrate, no formation of glyceraldehyde 3-phosphate could be observed.
Additionaly, it was shown that the glyceraldehyde 3-phosphates are instable under pH-conditions the enzymes are working. Therefore, the enzyme concentrations are also a critical parameter to achieve fast reaction rates.
Based on the unexpected behaviour of glyceraldehyde 3-phosphate at different pH-values, several related phosphorylated metabolites have been investigated regarding their pH stability.
1 Kalyananda, M. et al., Journal of Bacteriology, 1987, 169, 2488-2493
2 Desai, K.K., et al., Biochemistry, 2008, 47, 7983-7985
3 Tang, C.T. et al., Antimicrob.Agents Chemother., 1977, 11, 147-153
4Wong, C.-H. et al., Journal of Organic Chemistry, 1983, 48, 3199-3205
5Hayashi, S.-I. et al., Journal of Biological Chemistry, 1967, 242, 1030-1035
