ATP-dependent phosphorylation of alpha-substituted carboxylic acids catalyzed by pyruvate kinase.

Pyruvate kinase from rabbit muscle catalyzes an ATP-dependent phosphorylation of glycolate to yield 2-phosphoglycolate (F. J. Kayne (1974) Biochem. Biophys. Res. Commun. 59, 8-13). An investigation of anologous reactions with other alpha-substituted carboxylic acids reveals several new substrates for such a phosphorylation reaction. Thus the alpha-hydroxy carboxylic acids L-lactate, D-lactate, DL-alpha-hydroxybutyrate, DL-alpha-hydroxyvalerate, L-glycerate, D-glycerate, DL-nitrolactate, and DL-beta-chlorolactate are phosphorylated on the alpha-hydroxy group to give the corresponding phosphoesters. Thioglycolate is also a slow substrate for phosphorylation of the thiol group to give the phosphothioglycolate, and DL-thiolactate is phosphorylated in a very slow reaction to give phosphothiolactate. beta-Hydroxypyruvate is a substrate; but, unlike the reaction with pyruvate, with beta-hydroxypyruvate the equilibrium for the reaction lies in favor of ADP and the phosphorylated product which appears from 31P NMR data to be tartronate-semialdehyde-2-phosphate. 31P NMR spectroscopy has been used to verify the identity of the products for all of the reactions. Steady-state kinetic constants have been obtained for some of the more rapid reactions. The reactions with glycolate, L-glycerate, and beta-hydroxypyruvate have kcat values that are close to that for phosphorylation of pyruvate in the reverse of the physiological reaction.