Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp

Abstract

The adenylate cyclase in T. brucei is located in the plasma membrane. Partial kinetic analysis of the enzyme properties revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4 mM. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. At high concentrations, Mg2+ inhibited the enzyme. The enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than 1 binding site for Mg2+ on the enzyme. A study of the patterns of product inhibition revealed little or no effect of 3'':5''-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). The substrate-binding domain on T. brucei adenylate cyclase probably interacts mainly with the triphosphate portion of the ATP molecule. Enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline [epinephrine], adenosine, GTP and guanyl-5''-yl imidodiphosphate. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was inhibited by relatively high concentrations of Ca2+ (1 mM).