The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Abstract

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 2 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 (EC 50 ) 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 EC 50 for forskolin to maximally activate enzyme activity with or without G s␣ . 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-1015 to Ser mutation.The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases 1 and guanylyl cyclases (1).All nine isoforms have a similar structure, including two intensely hydrophobic domains (M 1 and M 2 ) and two 40-kDa cytoplasmic domains (C 1 and C 2 ). The two cytoplasmic domains contain sequences (C 1a and C 2a ) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein 2 ␣ subunit, designated as G s␣ , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein ␤␥ subunits, the ␣ subunit of G i , G o , and G z , Ca 2ϩ ion, Ca 2ϩ -calmodulin, Ca 2ϩ -calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. ␤␥) can be either stimulatory or inhibitory...

An Extended Conformation of Calmodulin Induces Interactions between the Structural Domains of Adenylyl Cyclase from Bacillus anthracis to Promote Catalysis

“…Interestingly, edema factor has a single DXD motif and we have shown that mutations at these two aspartates can drastically reduce catalysis (41). In addition, there is an arginine residue in mammalian adenylyl cyclase that is involved in the stabilization of a pentaphosphate intermediate (42,43). We have found that arginine 329 of edema factor is crucial for catalysis and may play a similar role in stabilizing the catalytic intermediate.…”

“…7). An arginine in mammalian adenylyl cyclase is implicated in stabilizing the pentaphosphate intermediate (42,43). We thus made and analyzed a mutant that had a mutation at one of the two conserved arginine residues, R329M (Fig.…”

Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment

“…The active site closure induced movements of a several active site residues conserved in all class III cyclases. The β7-β8 loop movement shifts Lys1184*, which in turn pushes Arg1150* of the neighboring α4 helix by 6 Å. Arg1150* is positioned by Asn1146* to be oriented toward the ATP ribose 3′ hydroxyl group and Pα such that it could stabilize the additional negative charge in the transition state 22 . The shift of the α1 helix rearranges the phosphate chain of the ATP analog, leading to a 180° flip of Pγ (Fig.…”

Searching for Neuronal Left/Right Asymmetry: Genomewide Analysis of Nematode Receptor-Type Guanylyl Cyclases

“…Both families contain a guanylyl cyclase catalytic domain ( Figure 2). In several GCY proteins, catalytically important residues are not conserved in the cyclase domain (Yan et al 1997) (supplemental Figure 1 at http://www.genetics.org/supplemental/) and it has been speculated that, in these cases, heterodimerization with a catalytically active GCY protein ensures activity of the dimer (Morton 2004). Apart from the presence of the cyclase domain, receptor-type and soluble GCY proteins differ significantly ( Figure 2).…”