Enzymatic synthesis and inhibitory characteristics of tartronate semialdehyde phosphate

The immediate product of the pyruvate kinase catalyzed phosphorylation of β-hydroxypyruvate is the enol of tartronate semialdehyde phosphate (TSP). The reaction has the same pH profile as that for the phosphorylation of pyruvate with pK's of 8.2 and 9.7 observed in H2O. This enol tautomerizes in solution to the aldehyde, which in turn becomes hydrated. 31P NMR spectra indicate that the enol resonates ∼ 1 ppm upfield from the hydrated aldehyde. By following the tautomerization spectrophotometrically at 240 nm, the authors have found it to be independent of pH, except that it is 2-fold slower above the pK of the phosphate group. It is 3.6-fold slower in D2O. When this TSP is reduced with NaBH4, ∼ 50% of the product is D-2-phosphoglyceric acid (substrate for enolase). Thus, while the immediate product of the phosphorylation reaction is the enol of TSP, the eventual product is D,L-TSP. Both the enol and the aldehyde forms of TSP were found to be potent inhibitors of yeast enolase with apparent Ki values of 100 nM and 5 μM, respectively. However, since the aldehyde form is 95-99% hydrated, the true Ki for the aldehyde species is 50-250 nM. The enol of TSP shows slow binding behavior, as expected for an intermediate analogue