The enzymatic equilibria of phospho(enol)pyruvate.

The reversibility of reaction (2) was discovered by Lardy and Ziegler (2), who showed that the failure of Meyerhof et al. (3) to demonstrate the backreaction with P32 was due to the absence of K ion. In the meantime it had been found by Boyer et al. (4) that K ion is indispensable for the transphosphorylation of phosphopyruvate with ADP. We have confirmed the results of Lardy and Ziegler, and have determined the equilibrium constant of reaction (2). Lardy’s mixture of triose phosphate, ATP, cozymase, inorganic phosphate, and phosphopyruvate or pyruvate, in the presence of dialyzed acetone powder extract of muscle can be used only for a demonstration of the reversibility of reaction (2), with or without P32. It does not, however, allow one to calculate the equilibrium constant, as has been erroneously assumed by some authors. If phosphopyruvate is synthesized from pyruvate by the ATP which forms continuously in the “coupling reaction” of glycolysis, the amounts formed depend on the time of reaction and the affinities of the various reactants in a complicated way; in particular, the relation of phosphopyruvate to pyruvate cannot be used for a calculation of the equilibrium constant. A determination of the equilibrium constant is only possible when the isolated reaction (2) is studied. In preliminary experiments we have used pyruvate labeled with Cl4 and measured the speed with which non-radioactive phosphopyruvate takes up Cl4 in the presence of dialyzed acetone