Gorren A C, Schrammel A, Schmidt K, Mayer B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-UniversitätGraz, Austria.
Biochemistry. 1997 Apr 8;36(14):4360-6. doi: 10.1021/bi962381s.
To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) we studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS. Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all bound to the heme with Kd values varying from 0.16 mM for DTT to 41 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorbance spectra with maxima at about 378 and 456 nm, indicative of bisthiolate complexes; the maximum at 426 nm with D-cysteine suggests binding of the neutral thiol. From the results with 2-mercaptoethanol we deduced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme ligand is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the same affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a > 100-fold lower affinity, indicative of competition between DTT and BH4 binding. Binding of DTT to the BH4-containing subunit was suppressed by L-arginine, whereas high-affinity binding was not affected, suggesting that L-arginine binds only to the BH4-containing subunit. DTT competitively inhibited L-citrulline production by nNOS containing one BH4 per dimer (Ki approximately 11 mM). Comparison of DTT binding and inhibition suggests that the heme of the BH4-free subunit is not involved in catalysis. Thermostability of nNOS was studied by preincubating the enzyme at various temperatures prior to activity determination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t1/2 approximately 6 min at 37 degrees C). At micromolar concentrations, inactivation was 10 times slower. Absorbance and fluorescence measurements demonstrate that inactivation was not accompanied by major structural changes. The stabilization of nNOS by thiols was illustrated by the fact that omission of 2-mercaptoethanol during preincubation for 10 min at 30 degrees C led to an activity decrease of up to 90%.
为了阐明硫醇如何影响神经元型一氧化氮合酶（nNOS），我们研究了硫醇与无四氢生物蝶呤（BH4）的nNOS的结合情况。二硫苏糖醇（DTT）、2-巯基乙醇以及L-和D-半胱氨酸均与血红素结合，其解离常数（Kd）值从DTT的0.16 mM到L-半胱氨酸的41 mM不等。DTT、2-巯基乙醇和L-半胱氨酸产生的吸收光谱在约378和456 nm处有最大值，表明形成了双硫醇盐配合物；D-半胱氨酸在426 nm处的最大值表明中性硫醇的结合。根据2-巯基乙醇的实验结果，我们推断在无2-巯基乙醇、无BH4的nNOS中，第六个血红素配体不是硫醇盐。DTT与每个二聚体含有一个BH4的nNOS的结合是双相的。显然，无BH4的亚基与DTT的结合亲和力与无BH4的酶相同，而含BH4的亚基的亲和力则低100倍以上，这表明DTT与BH4的结合存在竞争。L-精氨酸可抑制DTT与含BH4亚基的结合，而高亲和力结合不受影响，这表明L-精氨酸仅与含BH4的亚基结合。DTT竞争性抑制每个二聚体含有一个BH4的nNOS产生L-瓜氨酸（抑制常数Ki约为11 mM）。DTT结合与抑制作用的比较表明，无BH4亚基的血红素不参与催化作用。通过在活性测定前将酶在不同温度下预孵育来研究nNOS的热稳定性。在纳摩尔浓度下，nNOS在20℃时稳定，但在较高温度下会迅速失活（在37℃时半衰期约为6分钟）。在微摩尔浓度下，失活速度慢10倍。吸光度和荧光测量表明，失活过程中没有伴随主要的结构变化。硫醇对nNOS的稳定作用体现在以下事实上：在30℃预孵育10分钟期间省略2-巯基乙醇会导致活性降低高达90%。
