The "phosphoryl-enzyme" from phosphoglycerate kinase. Appendix: Crystalline 3-phospho-D-glycerate kinase from horse muscle

Abstract

The "phosphoryl-enzyme" prepared from yeast and horse muscle phosphoglycerate kinase [EC 2.7.2.3] ATP in the presence of an ADP trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably a tight complex between 1,3-bisphosphoglycerate and the enzyme. The 2 partial exchange reactions (between ADP and ATP, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are much slower than the rate of overall catalysis. No substrate analog accelerated the partial exchange reactions. Catalysis of each of the 2 exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.