The addition and release of magnesium in the phosphoglucomutase reaction. I. Evidence for a random order of addition.

Rate constants for release of Mg +, glucose -phosphate, glucose 6-phosphate, and glucose 1 ,6-diphosphate from the catalytically active complex under initial velocity conditions together with rate constants for dissociation of the Mg 2+ complexes of phosphoand dephosphoenzyme are used to define the order of addition and release of the above species during the phosphoglucomutase reaction. Although the reaction pathway is technically random, it is actually ordered kinetically to a very high degree. The difference between the concept of ordered versus random reaction pathways in enzymic reactions may thus be mainly a quantitative rather than a qualitative consideration. All-or-none type assays are described for measuring some of the above rate constants and for evaluating the dissociation constant of the phosphoenzyme-magnesium complex. The possibility of phosphate isomerization in the phosphoenzyme magnesium complex was examined. Since product inhibition in the phosphoglucomutase reaction is strictly competitive at high concentrations of product, kinetic data give no support to the possibility that phosphate transfer between two different attachment sites on the enzyme occurs during the catalytic cycle. Kinetic constants measured previously were identified in terms of the appropriate dissociation steps. The rate constants for release of Mg 2+ from the catalytically active enzyme substrate Mg complex, the phosphoenzymeMg complex, and the dephosphoenzyme-Mg complex are about 0.03, 0.07, and 0.22 sec, respectively; the release of Mg from the catalytically active complex is thus about 1/25,000 of the catalytic conversion of glucose-l-P to glucose-6-P, 750 sec-.