Effect of partial tryptic proteolysis on the kinetic properties of rabbit muscle glycogen synthase

Abstract

The kinetic basis for the changes in the activity of rabbit muscle glycogen synthase I (UDPglucose:glycogen 4α-glucosyltransferase, EC 2.4.1.11) following limited tryptic proteolysis has been investigated. The action of trypsin (0.63 μg/ml) caused a degradation of the native 85 000 dalton subunit to a species of 75 000 daltons and finally of 68 000 daltons. After 6 min of proteolysis, the percent I activity had fallen from 86% to 40% and the total activity (measured in the presence of glucose-6- P) by 30%. This corresponded to an enzyme composed primarily (75%) of the 68 000 dalton species and to a lesser extent (24%) of the 75 000 dalton species. The native 85 000 dalton subunit was almost totally absent. The kinetic properties of such tryptically degraded enzyme were compared with the native species: tryptic hydrolysis caused an increase in the S 0.5 for UDPglucose from 1 mM to 11 mM, and of the M 0.5 for glucose-6- P from 16 μM to 53 μM. This compares with values of 75 mM ( S 0.5) and 340 μM ( M 0.5), respectively, for glycogen synthase D form. Proteolysis also caused changes in the nature of the dependence of reaction rate on UDPglucose concentration, leading to a decrease in the Hill slope. The shape of the Hill plot for glucose-6- P variation, however, which was non-linear for the native enzyme, was unchanged by proteolysis. Whatever underlies this complex kinetic behaviour, then, must persist after proteolysis; this might mean, for example, that subunit interactions remained even in the degraded enzyme. The results demonstrate clearly that the kinetic parameters most strongly influenced by tryptic hydrolysis are the apparent affinities for the substrate UDPglucose and the activator glucose-6- P. Importantly, however, even extensive proteolysis is less effective than covalent phosphorylation in changing kinetic properties.