Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase.
Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase.
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L-精氨酸和咪唑的结合表明大鼠脑神经元一氧化氮合酶的异质性。
DOI：
10.1021/bi025675o
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
B. Mayer
中科院分区：
文献类型：
--
作者：
A. Gorren;K. Schmidt;B. Mayer
一氧化氮合酶（NOS）被咪唑抑制，咪唑在428 nm处吸收的低自旋复合物中与血红素结合。通过L-精氨酸将该复合物转化为在395 nm处吸收的高自旋物质是确定Arg结合参数的常用方法。然而，精氨酸竞争性和非竞争性抑制NOS的咪唑已被报道，光学研究与神经元NOS提供没有证据咪唑影响精氨酸结合。我们研究了重组大鼠脑神经元NOS这些矛盾的观察结果的原因。咪唑与nNOS结合的K（d）（app）为50 μ M;四氢生物蝶呤（BH 4）使nNOS对咪唑的亲和力降低4倍。该酶的行为与精氨酸结合不均匀。大多数nNOS（65-80%）表现为精氨酸与咪唑竞争。在BH 4存在下，该组分的K（d）（Arg）估计为1 μ M，表观缔合和解离速率常数分别为2.5 × 10（6）M（-1）× s（-1）和2.5 s（-1）。nNOS的第二部分（20-30%）表现出很少或没有竞争。因此，精氨酸结合并没有导致解离的咪唑复合物的这一部分，并完全生成的高自旋态的精氨酸不能实现在咪唑的存在下。第三部分（<或=10%）以低亲和力（K（d）1-2 mM）结合Arg。由于这种异质性，Arg的滴定曲线几乎无法解释。我们建议，这种异质性的nNOS对精氨酸和咪唑的反应是潜在的明显矛盾的结果在文献中报道。
Nitric oxide synthase (NOS) is inhibited by imidazole, which binds to the heme in a low-spin complex absorbing at 428 nm. Conversion by L-arginine of this complex into a high-spin species absorbing at 395 nm is a common method to determine the binding parameters of Arg. However, both Arg-competitive and noncompetitive inhibition of NOS by imidazole has been reported, and optical studies with neuronal NOS provided no evidence for imidazole affecting Arg binding. We investigated the cause for these paradoxical observations with recombinant rat brain neuronal NOS. Imidazole bound to nNOS with a K(d)(app) of 50 microM; tetrahydrobiopterin (BH4) lowered the affinity of nNOS for imidazole 4-fold. The enzyme behaved heterogeneously with respect to Arg binding. Most of nNOS (65-80%) showed competition between Arg and imidazole. In the presence of BH4, a K(d)(Arg) of 1 microM could be estimated for this fraction, as well as apparent association and dissociation rate constants of 2.5 x 10(6) M(-1) x s(-1) and 2.5 s(-1). A second fraction of nNOS (20-30%) exhibited little or no competition. Consequently, Arg binding did not cause dissociation of the imidazole complex for this fraction, and complete generation of the high-spin state by Arg could not be achieved in the presence of imidazole. A third fraction (< or =10%) bound Arg with low affinity (K(d) 1-2 mM). Because of this heterogeneity, titration curves with Arg became almost uninterpretable. We propose that this heterogeneous response of nNOS toward Arg and imidazole is underlying the apparently conflicting results reported in the literature.