Effectors of purified adenyl cyclase from Streptococcus salivarius

The activity of highly purified (3200-fold) adenyl cyclase from Streptococcus salivarius was examined in the presence and absence of P_i, inorganic pyrophosphate, nucleotides and glycolytic intermediates. The enzyme was inhibited by P_i, ADP and all the triphosphates and monophosphates of guanosine, uridine, inosine and cytidine; inhibition was completely competitive. The inhibition in the presence of PP_i was partially competitive, while AMP produced inhibition of the mixed type, i.e., partially competitive and completely noncompetitive. PP_i was the most potent inhibitor (K_i = 0.23 mm) followed by ADP (K_i = 0.43 mm), GTP (K_i = 0.52 mm) and UTP (K_i = 0.60 mm). Severe inhibition of the enzyme was also observed in the presence of the diphosphate and sugar nucleotides of the above bases at concentrations between 0.1 and 5 mm, when tested at one substrate concentration (ATP = 0.6 mm). The respective cyclic 3′,5′-nucleoside monophosphates were, however, only slightly inhibitory (maximum 11%).While the nucleotides were generally inhibitory, the activity of the enzyme was variable in the presence of various glycolytic intermediates. Weak activation of adenyl cyclase activity was observed with glucose-6-P, glucose-1-P, 2-P-glycerate and pyruvate. 2-P-glycerate required the lowest concentration for half maximal activation (K_a = 0.13 mm) followed by glucose-1-P (0.24 mm), glucose-6-P (0.55 mm) and pyruvate (1.12 mm). These compounds increased the V of the enzyme without affecting the apparent K_m for ATP. Fructose-6-P, fructose-1,6-P₂, glyceraldehyde-3-P and P-enolpyruvate inhibited or activated the enzyme depending upon the concentration of the compound used. Both the apparent K_m for ATP as well as the V were altered by fructose-6-P and fructose-1,6-P₂. However, activating concentrations of glyceraldehyde-3-P and P-enolpyruvate increased the V without affecting the apparent K_m, whereas inhibiting concentrations decreased the V and increased the K_m for the substrate.