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首页> 外文期刊>Biochemistry >Kinetic Characterization of Thiolate Anion Formation and Chemical Catalysis of Activated Microsomal Glutathione Transferase
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Kinetic Characterization of Thiolate Anion Formation and Chemical Catalysis of Activated Microsomal Glutathione Transferase

机译：硫氰酸根阴离子形成的动力学表征和活化的微粒体谷胱甘肽转移酶的化学催化

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Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated,up to 30-fold,by the reaction with sulfhydryl reagents,e.g.,N-ethylmaleimide.Analysis of glutathione (GSH) thiolate formation,which occurs upon mixing activated MGST1 with GSH,reveals biphasic kinetics,where the rapid phase dominated at higher GSH concentrations.The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K_D~(GSH)approx=10 mM),followed by slower formation of thiolate (k_2approx=10 s~(-1)).The release rate (or protonation of the enzyme GSH thiolate complex) of GS~- was slow (k_(-2)=0.016 s~(-1)),consistent with overall tight binding of GSH.Electrophilic second substrates react rapidly with the E centre dot GS~- complex,and again,a two-step mechanism is suggested.In comparison to the unactivated enzyme [Morgenstern et al.(2001) Biochemistry 40,3378-3384],the mechanisms of GSH thiolate formation and electrophile interaction are similar;however,thiolate anion formation is enhanced 30-fold in the activated enzyme,contributing to an increased k_(cat) (3.6 s~(-1)).Interestingly,in the activated enzyme,thiolate formation and proton release from the enzyme are not strictly coupled,because proton release (as well as k_(cat)) was found to be approx=4 times slower than GSH thiolate formation in an unbuffered system.Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k_2) for thiolate formation and k_(cat) (in the reaction with l-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1.This indicates that thiolate formation contributes to k_(cat) for the activated enzyme,as suggested previously for unactivated MGST1.The stoichiometry of thiolate formation,proton release,and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.
机译：微粒体谷胱甘肽转移酶1（MGST1）具有与巯基试剂（例如N-乙基马来酰亚胺）反应的独特能力，可被激活多达30倍。分析了将谷胱甘肽（GSH）硫醇盐的形成情况，是将活化的MGST1与GSH揭示了双相动力学，其中快速相在较高的GSH浓度下占主导地位。动力学行为表明，有一个两步机理，包括一个快速的GSH结合步骤（K_D〜（GSH）大约= 10 mM），随后是较慢的GSH结合过程。硫醇盐（k_2approx = 10 s〜（-1））。GS〜-的释放速率（或GSH硫醇盐复合酶的质子化）较慢（k _（-2）= 0.016 s〜（-1）），与亲电子的第二种底物与E中心点GS〜-络合物快速反应，并再次提出了两步机理。与未激活的酶相比[Morgenstern等人，（2001）Biochemistry 40,3378 -3384]，GSH硫醇盐的形成和亲电相互作用的机理相似;但是，硫醇盐a离子在活化酶中的形成增加了30倍，导致k_（cat）（3.6 s〜（-1））增加。有趣的是，在活化酶中，硫醇盐的形成和质子从酶的释放没有严格耦合，因为发现在无缓冲系统中质子释放（以及k_（cat））比GSH硫醇盐的形成慢约4倍。溶剂动力学同位素效应测量表明，硫醇盐的速率常数（k_2）降低了2倍MGST1和​​未活化的MGST1的形成和k_（cat）（与1-氯-2,4-二硝基苯反应）。这表明硫醇盐的形成对活化酶的k_（cat）有所贡献，如先前对未活化的MGST1的建议。硫醇盐形成，质子释放和爆裂动力学的化学计量表明每个酶三聚体利用一个GSH分子。

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