STUDIES ON SH GROUPS OF PHOSPHORYLASE B REACTION WITH 5,5'-DITHIOBIS-(2-NITROBENZOIC ACID]

The reaction between 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and the SH groups of phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) has been investigated. At pH 6.8 and 25° approx. 2 SH groups, SH1, reacted very rapidly, 4 SH groups, SH11, reacted more slowly, and the remaining 10 SH groups, SH111, reacted extremely slowly with DTNB. The second order rate constants for the SH11 and SH111 groups were found to be 3.1·10-1 and 8.5·10-2 M-1·min-1, respectively, while the rate constant for the SH1 groups was estimated to be larger than 1·105 M-1·min-1. The loss of enzymatic activity paralleled the decrease in the number of free SH11 groups. Reaction with SH11 also resulted in dissociation of the enzyme into subunits with s20,w=5.6S. In the presence of sodium dodecyl sulfate approx. 16 SH groups reacted; the rate of reaction showed a maximum with respect to the concentration of sodium dodecyl sulfate, the molal ratio between sodium dodecyl sulfate and protein being approx. 3.5·102 at the maximum. AMP protected the SH11 groups but not SH1 or SH111 groups. Glycogen and glucose 1-phosphate, on the other hand, had little effect. The reactivity of the SH11 groups increased in the presence of salt, whereas the number of rapidly reacting SH groups was found to depend on the pH of the reaction mixture and reached a minimum at approximately pH 7. The reactivity of the SH groups on apophosphorylase b was found to be quite different from that of the holoenzyme. 6 SH1 type groups were detected on the apoenzyme, and in the presence of AMP this number increased to approx. 8. The enzymatic activity of the DTNB-treated enzyme could be fully restored by addition of reducing agents such as 2-mercaptoethanol. The reconstituted enzyme possessed the same sedimentation properties as the native holoenzyme. © 1969.