Effectors of purified adenyl cyclase from Streptococcus salivarius

Abstract

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 m m) followed by ADP ( K i = 0.43 m m), GTP ( K i = 0.52 m m) and UTP ( K i = 0.60 m m). 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 m m, when tested at one substrate concentration (ATP = 0.6 m m). 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 m m) followed by glucose-1-P (0.24 m m), glucose-6-P (0.55 m m) and pyruvate (1.12 m m). These compounds increased the V of the enzyme without affecting the apparent K m for ATP. Fructose-6-P, fructose-1,6-P 2, 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-P 2. 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.