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 activity, Mutational and genetic analyses of type I and soluble adenylyl cyclases 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 catalytic activity of the C-1 domain, Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C-2 domains, but not among the C-1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result 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 concentration (EC50) for the C-2 domain of type II adenylyl cyclase to associate with the C-1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without G(s alpha). This indicates that the mutations at these two residues do not cause gross structural alteration. 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 catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated, This suppressor has 17-fold higher activity than the mutant and has a Pro-101B to Ser mutation.