Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase
Nitric oxide-dependent allosteric inhibitory role of a second nucleotide binding site in soluble guanylyl cyclase
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DOI：
10.1074/jbc.m412203200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Beuve, A
中科院分区：
文献类型：
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作者：
Chang, FJ;Lemme, S;Beuve, A
一氧化氮（NO）受体(α(1)中心点β（1)可溶性鸟酰环化酶（sGC）异构体）脱敏的机制尚不清楚。基于腺苷酸环化酶（AC） x射线晶体结构的α(1)中心点β(1)结构模型表明，除了假定的催化位点外，还存在一个类似核苷酸的结合位点。我们之前报道过，将α(1)中心点β(1)中协调Mg2+ GTP（底物）结合的残基突变为AC中存在的残基，完全将GC活性恢复为AC活性。纯化α(1)中心点β(1)野生型（GC-wt）和突变型(AC-mut, α (1)R592Q中心点β (1)E473K, C541D)，并评估它们对各种核苷酸的敏感性。在使用AC-mut以及其他协调嘌呤结合的突变体时，我们能够区分鸟嘌呤核苷酸的变构抑制作用和对催化活性的竞争性抑制作用。在这里，我们报道了几个核苷酸类似物通过作用于第二个核苷酸位点（可能与催化位点假对称）而急剧改变sGC和AC-mut活性。特别是，Mg(2+)GTP γ S和Mg(2+)ATP γ S通过混合的非竞争性机制抑制环化酶活性，这种机制仅在NO刺激下观察到，而在基础条件下没有观察到。非竞争性抑制模式在携带替代β (1)D477A的突变体中不存在，与α (1)D529的假对称等效（位于底物结合位点，参与底物结合和催化），或具有双重突变α (1)E525K, C594D，与β (1)E473K， C541D的假对称等效。综上所述，这些数据表明，核苷酸占据第二位点可能是sGC脱敏的部分机制。
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.