Mechanism-Based Enzyme Inactivation Using an Allyl Sulfoxide-Allyl Sulfenate Ester Rearrangement1.
Mechanism-Based Enzyme Inactivation Using an Allyl Sulfoxide-Allyl Sulfenate Ester Rearrangement1.
复制标题
使用烯丙基亚砜-烯丙基亚磺酸酯重排进行基于机制的酶失活1。
DOI：
10.1021/ja00532a042
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发表时间：
1980
影响因子：
15
通讯作者：
Firestone,RaymondA
中科院分区：
文献类型：
--
作者：
Johnston,Michael;Raines,Ronald;Walsh,Christopher;Firestone,RaymondA
合成了2-氨基-4-氯-5-（p-硝基苯亚磺酰基）戊酸（1），并显示出诱导两种磷酸吡哆醛依赖性酶的基于机理的失活：（1）胱硫醚7-合成酶，其催化细菌甲硫氨酸生物合成中的7-取代反应;和（2）甲硫氨酸7-裂解酶，其催化细菌甲硫氨酸分解中的7-消除反应。失活是不可逆的，并显示饱和动力学。当被2-氨基-4-氯-5-（对硝基[3 H]苯基亚磺酰基）戊酸（Ia）灭活时，每种酶每摩尔酶单体掺入大约1摩尔氚，证实每种蛋白质的修饰是共价的和化学计量的。当甲硫氨酸7-裂解酶被2-氨基-4-氯-5-[3 H]-5-对硝基苯基亚磺酰基）戊酸（Ib）完全灭活时，给出亚化学计量标记（每摩尔酶单体0.12摩尔氚）。这两种酶，失活的1，是易于再活化的硫醇。失活的胱硫醚7-合成酶在与过量的二硫苏糖醇孵育后可恢复25%的催化活性，而甲硫醚7-裂解酶可被二硫苏糖醇、巯基乙醇和巯基丙酸酯100%再活化。再活化产生对硝基苯基硫醇阴离子，在甲硫氨酸7-裂解酶的情况下，与再活化的酶化学计量地形成。这两种酶在硫醇的存在下被1“保护”免于失活，其同时产生p-硝基苯基硫醇。在二硫苏糖醇存在下，对硝基苯硫酚的保护反应符合准一级反应动力学。2-氨基-4-氯-5-（叔丁基）-2-（三氟甲基）苯甲酸甲苯亚磺酰基）戊酸（2）和2-氨基-4-（p-硝基苯基亚磺酰基）-5-氯戊酸（3），1的反向区域异构体，也已经制备，并且没有给出任何一种酶失活的证据。这些数据表明了一种新的自杀失活形式（方案II），其中β-碳负离子辅助的7-卤化物消除产生烯丙基亚砜-酶-吡哆醛加合物（4），该加合物经历自发的2，3-σ迁移重排成亲电子的烯丙基亚磺酸酯（5）。后者然后被酶亲核试剂捕获，得到失活的酶6，其可能是混合的二硫化物，或者不太可能是次磺酰胺。
2-Amino-4-chloro-5-(p-nitrophenylsulfinyl) pentanoic acid (1) has been synthesized and shown toinduce mechanism-based inactivationof two pyridoxal phosphate dependent enzymes:(1) cystathionine 7-synthetase, which catalyzes a 7-replacement reaction in bacterial methionine biosynthesis; and (2) methionine 7-lyase, which catalyzes a 7-elimination re-action in bacterial methionine breakdown. The inactivations are irreversible and display saturation kinetics. Each enzyme incorporates roughly 1 mol of tritium per mol of enzyme monomer when inactivated by 2-amino-4-chloro-5-(p-nitro [3H] phenylsulfinyl) pentanoic acid (la), confirming that the modification of each protein is covalent and stoichiometric. Substoichiometric labeling (0.12 mol of tritium per mol of enzyme monomer) is given when methionine 7-lyase is fully inactivated by 2-amino-4-chloro-5-[3H]-5-p-nitrophenylsulfinyl) pentanoic acid (lb). Both enzymes, inactivated by 1, are susceptible to reactivation by thiols. Inactivated cystathionine 7-synthetase recovers 25% of its catalytic activity upon incubationwith excess dithiothreitol, while methinonine 7-lyase is 100% reactivated by dithiothreitol, mercaptoethanol, and mercaptopropionate. Reactivation gen-erates p-nitrophenylthiolate anion, which forms, in the case of methionine 7-lyase, stoichiometrically with enzyme reactivated. Both enzymes are “protected” from inactivation by 1 in the presence of thiols, which simultaneously generates p-nitrophenylthiol. Inthe presence of dithiothreitol, the protection reaction gives p-nitrophenylthiol production with pseudo-first-order kinetics. 2-Amino-4-chloro-5-(/?-tolylsulfinyl) pentanoic acid (2) and 2-amino-4-(p-nitrophenylsulfinyl)-5-chloropentanoic acid (3), the reverse regioisomer of 1, have also been prepared and give no evidence of inactivation of either enzyme. The data are taken to indicate a novel form of suicide inactivation (Scheme II) wherein ß-carbanion-assisted 7-halide elimination generates an allyl sulfoxide-enzyme-pyridoxal adduct (4) which undergoes spontaneous 2, 3-sigmatropic rearrangement to an electrophilic allyl sulfenate ester (5). The latter is then captured by an enzymic nucleophile to give an inactive enzyme 6, which may be a mixed disulfide or, less likely, a sulfenamide.