Kinetic mechanism of potato phosphorylase.

Polysaccharide phosphorylase isolated from Idaho potatoes was the subject of a steady state kinetic study. The initial velocity of the reaction between a-D-glucopyranose l-phosphate (glucose-l-P) and amylopectin, in the absence of orthophosphate, was determined as a function of substrate concentration. A similar study was carried out for the reverse reaction. The result is characteristic of a sequential reaction mechanism in which both substrates must add to the enzyme before any product can be liberated. Isotope exchange rates at chemical equilibrium were determined for the glucose-1-P:Pi: amylopectin system in the presence of phosphorylase. Exchange of 32P from glucose-1-P into Pi and exchange of 14C from glucose-l-P into amylopectin were followed simultaneously by the use of glucose-l-F containing both isotopes. Concentrations of glucose-l-P and Pi were varied together in their equilibrium ratio at constant amylopectin concentration, and the concentration of amylopectin was varied at fixed concentrations of phosphates. Exchange rates for the two isotopes were equal under all conditions and gave linear reciprocal plots. These results support a rapid equilibrium mechanism. Isotope exchange rates were also determined under nonequilibrium conditions. Exchange of 3ZP from glucose-l-P into Pi was followed as a function of amylopectin concentration at a fixed concentration of glucose-l-P and several fixed concentrations of PiThe exchange was also followed as a function of glucose-l-P concentration at a fixed amylopectin concentration and several fixed Pi concentrations. These experiments are equivalent to conventional product inhibition experiments. The results indicate that Pi is a competitive inhibitor with respect to glucose-l-P and a noncompetitive inhibitor with respect to amylopectin. Our conclusion is that potato phosphorylase has a rapid equilibrium Random Bi Bi mechanism (CLELAND, W. W.,