The conserved asparagine and arginine are essential for catalysis of mammalian adenylyl cyclase.
Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha. 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 C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat 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-1015 to Ser mutation.
Yan
S Z
SZ
Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637, USA.
Huang
Z H
ZH
Shaw
R S
RS
Tang
W J
WJ
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001216:Asparagine; D004252:DNA Mutational Analysis; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012995:Solubility; D013329:Structure-Activity Relationship
D001216:Asparagine; D001120:Arginine; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase