Pyruvate, phosphate dikinase. Metal cation requirements and inactivation of the enzyme by sulfhydryl agents.

The metal cation requirements of pyruvate, phosphate dikinase from Bacteroides symbiosus have been examined with respect to both the overall reaction and the three partial reactions. The overall reaction and all three partial reactions require a divalent cation (M$‘, Mn’+, or Co’+). Although Mn2+ and Co’+ are very effective activators at low concentrations in two of the three partial reactions, they each inhibit one partial reaction at higher concentrations. M$+ is not inhibitory to any of the partial reactions and thus is the most effective divalent ion activator for the complete overall reaction. The monovalent cation requirement of the dikinase (Tl’, N&+, or K+) was found to reside completely in the P-enolpyruvate, pyruvate partial reaction, which is consistent with the involvement of monovalent cations in enolization reactions. Fully purified dikinase possesses a maximum of 18 sulfhydryl groups/dimeric molecule of which two or three are essential for enzymatic activity. The number of sulfhydryl groups decreased during loss of enzyme activity with aging. The relative reactivity of the sulfhydryl groups of the native enzyme (IQ, phosphorylated enzyme (I&), and pyrophosphorylated enzyme (I&,) was compared. The sulfhydryl groups of Z$, react with 5,5-dithiobis(2-nitrobenzoic acid) (D!l’NB) at a higher rate than those of either E or Epp with a concomitant increased rate of loss of enzyme activity. However, Ep in the form of the transition state analog complex (consisting of E,, Me’+, oxalate, and an activating monovalent cation) shows a decreased reactivity of the sulfhydryl groups and decreased inhibition of the enzyme by DTNB. Apparently, a conformation change occurs during formation of the complex which protects the essential sulfhydryl groups. Dikinase is dimeric and exhibits some properties of half-site reactivity. The sulfhydryl groups of the diphosphorylated enzyme (Gp) were found to be more reactive with DTNB than those of the monophosphorylated enzyme (E,) but the sulfhydryl groups of the transition state analog complex of pEp were less reactive and the enzymatic activity was less subject to inactivation by DTNB. Apparently, when only one subunit of the enzyme is phosphorylated, the nonphosphorylated subunit may still react with DTNB.