Studies on the SH groups of phosphorylase b Reaction with 5,5′-dithiobis-(2-nitrobenzoic acid)

Abstract

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, SH 1, reacted very rapidly, 4 SH groups, SH 11, reacted more slowly, and the remaining 10 SH groups, SH 111, reacted extremely slowly with DTNB. The second order rate constants for the SH 11 and SH 111 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 SH 1 groups was estimated to be larger than 1·10 5 M -1·min -1. The loss of enzymatic activity paralleled the decrease in the number of free SH 11 groups. Reaction with SH 11 also resulted in dissociation of the enzyme into subunits with s 20, 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. 3.5·10 2 at the maximum. AMP protected the SH 11 groups but not SH 1 or SH 111 groups. Glycogen and glucose 1-phosphate, on the other hand, had little effect. The reactivity of the SH 11 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 SH 1 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.