KINETIC EFFECTS OF UREA ON ACTIVATION OF AGED GLYCOGEN PHOSPHORYLASE A BY ADENOSINE 5'-PHOSPHATE

Previous observations have indicated that the sigmoid curve of rate versus Pi concentration obtained with aged preparations of phosphorylase a (a-1,4-glucan; orthophosphate glucosyltransferase, EC 2.4.1.1), is abolished by the activator AMP. The effects of urea on the kinetics of the enzymatic reaction were investigated in order to help clarify the relationship between structure and function. Urea also abolished the sigmoid curve although at concentrations 10 000-fold higher than AMP. However, although 2·10-5M AMP reduced the apparent Km for Pi, 0.3 M urea was a competitive inhibitor with respect to Pi, and 1.0 M urea caused inhibition of the mixed type. Mixed inhibition experiments with urea and arsenate, a true competitor of Pi, and the reduction of the Km for AMP by both urea and arsenate indicated that urea indeed competed with Pi. Urea did not affect the V for AMP, whereas arsenate reduced it. The data indicated that AMP protected the enzyme against urea inhibition by reducing the affinity of the enzyme for urea. It is suggested that aging causes changes in the structure of the enzyme which allow easier demontration of homotropic interactions. © 1969.