Mechanism and kinetics of inducible nitric oxide synthase auto-S-nitrosation and inactivation.
Mechanism and kinetics of inducible nitric oxide synthase auto-S-nitrosation and inactivation.
复制标题
诱导型一氧化氮合酶自动 S-亚硝化和失活的机制和动力学。
DOI：
10.1021/bi201818c
复制
发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Marletta,MichaelA
中科院分区：
文献类型：
--
作者：
Smith,BrianC;Fernhoff,NathanielB;Marletta,MichaelA
一氧化氮（NO）是一氧化氮合酶（NOS）反应的产物，以前的研究表明，一氧化氮合酶（NOS）会导致Zn2+-四硫酸盐的s -亚硝化和酶的失活。为了探讨noss -亚硝化的潜在生理意义，我们测定了诱导型NOS亚型（iNOS）的失活时间尺度，发现其与noss -亚硝化水平的升高直接相关。建立了以精氨酸作为自杀底物的NOS失活动力学模型。在该模型中，血红素辅助因子合成的NO被分为释放到溶液中（NO释放途径）和NOS -亚硝化然后NOS失活（失活途径）。实验确定的NO生成过程曲线与模型拟合。通过添加NO陷阱氧肌红蛋白（MbO2）和β2 H-NOX干扰NO释放途径，在4 μM MbO2和饱和陷阱浓度下，NO释放和失活之间的分配比为~ 100和~ 22000。结果表明，在血红素辅助因子处合成的一部分NO与Zn2+-四硫酸盐发生反应而不释放到溶液中。通过添加还原剂GSH或TCEP对失活途径进行扰动，导致inoss -亚硝化水平呈浓度依赖性降低，这与保护iNOS失活直接相关。NOS的失活对GSH生理浓度的响应最大，表观km值为13 mM。NOS的转换导致noss -亚硝化可能是控制NOS活性的机制，noss -亚硝化可能在亚硝基硫醇的生理生成中起作用。
Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result inS-nitrosation of the NOS Zn2+-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOSS-nitrosation, we determined the inactivation time scale of the inducible NOS isoform (iNOS) and found it directly correlates with an increase in the level of iNOSS-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOSS-nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO2) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ∼100 at 4 μM MbO2and ∼22000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn2+-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agent GSH or TCEP resulted in a concentration-dependent decrease in the level of iNOSS-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparentKmvalue of 13 mM. NOS turnover that leads to NOSS-nitrosation might be a mechanism for controlling NOS activity, and NOSS-nitrosation could play a role in the physiological generation of nitrosothiols.