Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase

The mechanism of desensitization of the nitric oxide (NO) receptor (alpha(1)center dot beta(1) isoform of soluble guanylyl cyclase, sGC) is not known. Models of the structure of alpha(1)center dot beta(1), based on the x-ray crystal structure of adenylyl cyclase (AC) suggest the existence of a nucleotide-like binding site, in addition to the putative catalytic site. We have previously reported that mutating residues that coordinate Mg2+ GTP (substrate) binding in alpha(1)center dot beta(1) into those present in AC fully reverts GC activity to AC activity. The wild-type form of alpha(1)center dot beta(1) (GC-wt) and the mutant form (AC-mut, alpha(1)R592Q center dot beta(1)E473K, C541D) were purified, and their sensitivities to various nucleotides were assessed. In using the AC-mut as well as other mutants that coordinate purine binding, we were able to distinguish allosteric inhibitory effects of guanine nucleotides from competitively inhibitory effects on catalytic activity. Here we report that several nucleotide analogs drastically alter sGC and AC-mut activity by acting at a second nucleotide site, likely pseudosymmetric to the catalytic site. In particular, Mg(2+)GTP gamma S and Mg(2+)ATP gamma S inhibited cyclase activity through a mixed, non-competitive mechanism that was only observable under NO stimulation and not under basal conditions. The non-competitive pattern of inhibition was not present in mutants carrying the substitution beta(1)D477A, the pseudosymmetric equivalent to alpha(1)D529 (located in the substrate-binding site and involved in substrate binding and catalysis), or with the double mutations alpha(1)E525K, C594D, the pseudosymmetric equivalent to beta(1)E473K, C541D. Taken together these data suggest that occupation of the second site by nucleotides may underlie part of the mechanism of desensitization of sGC.