The methylene analogue of 3-phospho-d-glycerate, 2-hydroxy-4-phosphono-dl-butyric acid, is a substrate for phosphoglycerate kinase. The pKa values for the final dissociation of the natural substrate and its methylene isostere are 6.20 and 7.45 respectively. The kinetic parameters Km and kcat. for the enzyme-catalysed reaction were determined at pH6.9 and 8.5 by using low substrate concentrations. Although the kcat. values for the two substrates at each pH are similar, there is a 60-fold increase in the Km value for the methylene isostere on going to the lower pH. The results are most readily interpreted in terms of a dianionic group on C-3 being required for efficient substrate binding to the enzyme.
