Secondary isotope effect in the phosphorylase reaction

The mechanism of action of phosphorylase B was studied by means of both α deuterium and α‐tritium secondary kinetic isotope effects of the enzymatic reaction. Two pairs of labelled substrates were synthesized : [1‐ 3 H]glucose 1‐phosphate, [U‐ 14 C]glucose 1‐phosphate and [1‐ 14 C, 1‐ 2 H]glucose‐1‐phosphate with [6‐ 3 H]glucose 1‐phosphate. The measurements showed that the isotope effects of the enzymatic synthesis of glycogen as well as of the reverse reaction of glycogen phosphorolysis were equal to zero. The secondary α‐tritium isotope effect of the acid hydrolysis of glucose‐1‐phosphate was estimated. The ratio K H / K 3H in this case was found to be 1.21. These data are consistent with the mechanism of direct displacement at the C‐1 atom of glucose and the formation of an intermediate enzyme · glucose‐1‐phosphate complex depending on the presence of glycogen.