The role of -SH groups in the enzymic activity of phosphorylase-b

Abstract The effect of 5′-AMP and substrate-constituents on the inactivation of phosphorylase-b caused by -SH reagents (PCMB and NEM) was investigated. It was found that in the case of PCMB-inactivation, glucose-1-phosphate increases and 5′-AMP decreases the inactivation rate, while glycogen has only a slight protecting effect. The same effects could be observed also in the NEM-inactivation experiment, except the protecting effect of 5′-AMP was smaller. Glycogen or 5′-AMP relieves the sensitizing effect of glucose-1-phosphate. 5′-AMP in combination with glucose-1-phosphate or glycogen protects more effectively than by itself. It was suggested that some of these effects of substrates and activator can be explained by conformation change of the enzyme. While 5′-AMP, especially in the presence of substrate, protects the -SH groups, it was found that this influence is mutual: the integrity of -SH groups assures the 5′-AMP binding of phosphorylase as was estimated by kinetic measurements. -SH compounds such as cysteine or mereaptoethanol decrease the Km value for 5′-AMP, while PCMB-treated phosphorylase has a greater Km for 5′-AMP than the control enzyme.