Properties of the highly reactive SH groups of phosphorylase b

The reaction of rabbit muscle phosphorylase b with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied with stopped-flow spectrophotometry. Two highly reactive sulfhydryl groups per dimer reacted with Nbs2 within a few seconds, while the remaining SH groups needed several minutes and hours. Decomposition of the time curve revealed that the highly reactive SH groups can be divided into two subclasses: a fast type which reacted with a rate constant of 3 x 10-3 M-1 sec-1 and a more slowly reacting type disappearing with a rate constant of 0.3 x 10-3 M-1 sec-1. The reactivity of the slowly reacting type increased by a factor of about 2 in the presence of 1 mM AMP. Concurrently, the ratio between the fast reacting and the more slowly reacting subclasses decreased from 5.3 to 1.0. The AMP effect was greatly enhanced by glucose 1-phosphate. This enhancement was abolished in the presence of ATP. The finding that the ratio between the number of SH groups in the two subclasses of the highly reactive SH groups changed upon addition of ligand molecules indicates that the two subclasses reflect the different reactivities of the SH groups when the enzyme is present in different conformational states. It is suggested that the highly reactive SH group measured belong to the peptide: Gly-Cys-Arg-Asp.