Purification and Properties of Adenyl Cyclase from Streptococcus salivarius

Adenyl cyclase has been purified from the soluble fraction of Streptococcus salivarius by differential centrifugation, DNase treatment, ammonium sulfate fractionation, and gel filtration on Sephadex G-200. The enzyme was repeatedly recovered from the Sephadex G-200 column in three distinct peaks, each devoid of ATPase, GTPase, cyclic adenosine 3',5'-monophosphate phosphodiesterase, cyclic guanosine 3',5'-monophosphate phosphodiesterase, and pyrophosphatase. The enzyme in all peaks (I, II, III), however, contained a small (2.1 to 6.7%) amount of guanyl cyclase activity. Enzyme III was the most homogeneous, containing only two faint contaminating bands on disc gel electrophoresis. The enzyme was Mg2+- or Mn2+-dependent, was stabilized by cysteine (5 and 10 mm) and was activated by NaF. While the pH optimum for Enzymes II and III was 8.0, that for Enzyme I was pH 7.5. The Km values for Enzymes I and II were both 1 mm for ATP, and were unaffected by 20 mm NaF, the optimal concentration for activation of the enzyme. The Km value for Enzyme III, however, was increased from 0.7 to 3.3 mm in the presence of NaF. In all cases, the Vmax for all three enzyme preparations was increased in assays with NaF by as much as 5-fold (e.g. II and III). Hill plots for all enzyme preparations gave slope values (n) of 1 indicating only one substrate-binding site on the enzyme; the slopes were unaffected by NaF. Sulfhydryl inhibitors, N-ethylmaleimide, and p-hydroxymercuribenzoate, were almost completely inhibitory at 1 mm suggesting the involvement of —SH groups in catalysis. Cysteine (1 mm) limited inhibition by these reagents significantly at inhibitor concentrations below 0.1 mm. ATP synthesis from cyclic adenosine 3',5'-monophosphate and pyrophosphate by adenyl cyclase was favored with K values for ATP formation ranging from 43 to 64. NaF inhibited the reverse reaction, with the reduction in reaction rate most severe with Enzyme III (73%). The average ΔF° for the synthesis of ATP was -2.48 kcal per mole.