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

The adenylyl and guanylyl cyclases catslyze 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 soluble cyclase of dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanyly 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 1 adenylyl cyclase coordinate a Mg(2+)ion. The D310S and D310A mutants have lO-fold reduced V-max and altered [Mg2+] 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 guanyly 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.