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

Abstract

The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two 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 well 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.

Sequence and structure of yeast phosphoglycerate kinase.

“…experiments showed that similar enzyme preparations contained significant amounts of organic phosphate (single phosphoryl peak) before, but not after, exhaustive dialysis. This correlates with the finding of Johnson et al (1976) that preparations of yeast PGK can contain contaminating amounts of a triose substrate. Conroy et al (1981) have since shown that active site arginine residues react only slowly with phenylglyoxal unless PGK is first incubated with the enzyme and cofactors which are required to convert 3-PGA to glyceraldehyde-3-phosphate.…”

“…Although both enzymes bind 3-PGA the reactions catalysed are very different. The mutase forms a phosphoryl intermediate (Rose, 1980) whereas the kinase does not (Johnson et al, 1976).…”

Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

“…Other nucleoside-metabolizing enzymes have not exhibited a similar preference for Ldeoxynucleoside analog diphosphates, and the property is unique to PGK. Human PGK (ϳ46 kDa) is a glycolytic enzyme that catalyzes the conversion of 1,3-biphosphoglycerate to 3-phosphoglycerate and during the process generates one molecule of ATP (17). The reaction is reversible.…”

Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase

“…Also, the isoelectric point of the renatured enzyme focused in 3M-urea was identical with that of native enzyme, also focused in 3 M-urea. Such an anion could be the substrate for the enzyme, 1,3-diphosphoglycerate, which Johnson et al (1975Johnson et al ( , 1976) have shown to be tightly bound to the enzyme.…”