Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis

The adenylyl and guanylyl cyclases catalyze the formation of 3′,5′-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5′-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C ₂ catalytic domain was used to model by homology a mammalian adenylyl cyclase C ₁ -C ₂ domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum , a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg ²⁺ ATP or Mg ²⁺ GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg ²⁺ ion. The D310S and D310A mutants have 10-fold reduced V _max and altered [Mg ²⁺ ] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.