Phosphoryl Intermediates Formed with Phosphoglycerate Mutase: ROLE AND LABILIZATION OF 2,3-DIPHOSPHOGLYCERATE

Abstract

Abstract A complex formed between phosphoglycerate mutase from rabbit muscle and its cofactor, 2,3-diphosphoglycerate, has been isolated and examined. With a column of Sephadex G-25, enzyme-bound radioactivity equivalent to 0.85 mole of cofactor per mole of enzyme was separated from 2,3-diphosphoglycerate-32P. As much as 80% of the enzymebound 32P could be incorporated into nonradioactive substrate or cofactor during very short reaction periods. These exchange reactions suggested that the bound radioactivity was not in the form enzyme-phosphate, but rather in the form enzyme·P2·glycerate. Subjecting enzyme with bound 32P to paper chromatography confirmed this; the radioactivity was distributed about equally between orthophosphate and monophosphoglycerate. This enzyme-bound cofactor was labile to treatment with 0.4 m KOH or HClO4, and was then detected as 32Pi after paper chromatography. 2,3-Diphosphoglycerate alone was not hydrolyzed under the same conditions. Thus, in the process of binding to the enzyme, the glycerate-phosphate bonds of the cofactor are rendered labile. The complex was unstable at 4°, decomposing into Pi and, apparently, glyceric acid. In a 24-hour period, the exchangeable bound radioactivity decreased from 73 to 24%, while paper chromatography of the complex showed a decrease in the monophosphoglycerate fraction and an increase in the Pi fraction. Enzyme-bound 32P from 3-phosphoglycerate-32P was obtained by a similar procedure. The binding was equivalent to 0.34 mole of substrate per mole of enzyme. This bound radioactivity was labile to treatment with 0.4 m HClO4 or KOH. Most of the bound radioactivity could be exchanged with nonradioactive cofactor or substrate. Unlike the enzyme-cofactor complex, a high percentage of bound radioactivity was not exchanged, and was recovered as Pi in exchange reactions with high concentrations of combined unlabeled 3-phosphoglycerate and 2,3-diphosphoglycerate. Exchange between 3-phosphoglycerate-32P and nonradioactive 2,3-diphosphoglycerate in the presence of phosphoglycerate mutase showed that free 2,3-diphosphoglycerate is not an obligatory reactant in every conversion of substrate to product. This is consistent with a cofactor role for free 2,3-diphosphoglycerate of maintaining saturating levels of an active intermediate, enzyme·P2·glycerate.