THE CONSERVED ASPARAGINE AND ARGININE ARE ESSENTIAL FOR CATALYSIS OF MAMMALIAN ADENYLYL-CYCLASE

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C -1 and C-2), and both domains are required for the high enzymatic acti vity, Mutational and genetic analyses of type I and soluble adenylyl c yclases suggest that the C-2 domain is catalytically active and the C- 1 domain is not; the role of the C-1 domain is to promote the catalyti c activity of the C-1 domain, Two amino acid residues, Asn-1025 and Ar g-1029 of type II adenylyl cyclase, are conserved among the C-2 domain s, but not among the C-1 domains, of adenylyl cyclases with 12 putativ e transmembrane helices. Mutations at each amino acid residue alone re sult in a 30-100-fold reduction in K-cat of adenylyl cyclase. However, the same mutations do not affect the K-m for ATP, the half-maximal co ncentration (EC50) for the C-2 domain of type II adenylyl cyclase to a ssociate with the C-1 domain of type I adenylyl cyclase and achieve ma ximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without G(s alpha). This indicates that the m utations at these two residues do not cause gross structural alteratio n. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C-1 domains may account for its lack of catalytic activity, Mutations at both amino acid residues together result in a 3,000-fold reduction in K-cat of adenylyl cyclase , suggesting that these two residues have additive effects in catalysi s. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated, This suppressor has 17-fold higher activity than the mu tant and has a Pro-101B to Ser mutation.