Park O K, Bauerle R
Department of Biology, University of Virginia, Charlottesville, Virginia 22903-2477, USA.
J Bacteriol. 1999 Mar;181(5):1636-42. doi: 10.1128/JB.181.5.1636-1642.1999.
The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.
已发现来自大肠杆菌的苯丙氨酸敏感型3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸合酶[DAHPS(Phe)]在体外不稳定是由于金属催化的氧化机制。DAHPS(Phe)是该酶的三种受不同反馈调节的同工型之一，它催化芳香族生物合成的第一步，即由磷酸烯醇丙酮酸和D-赤藓糖-4-磷酸形成DAHP。脱辅基酶的活性呈指数衰减，在室温下半衰期约为1天，并且异源四聚体缓慢解离为单体状态。磷酸烯醇丙酮酸或EDTA的存在使该酶稳定，这表明在没有底物的情况下，微量金属是失活剂。Cu2+和Fe2+，但不是激活该酶的其他二价金属，极大地加速了失活速率和亚基解离。厌氧和添加过氧化氢酶均显著降低了Cu2+催化的失活。在自发失活的酶中，每个亚基的七个巯基中有两个净损失；这个值随着添加的Cu2+浓度增加而增加。二硫苏糖醇完全恢复了自发失活酶的酶活性和两个丢失的巯基，但对Cu2+失活酶的再激活仅部分有效。在两个活性位点半胱氨酸（Cys61或Cys328）之一处进行保守替换的突变酶对金属攻击不敏感。对Cu2+失活酶的肽图谱分析揭示了这两个半胱氨酸残基之间的二硫键连接。所有结果表明，DAHPS(Phe)是一种金属催化的氧化系统，其中结合的底物可保护活性位点残基免受结合的氧化还原金属辅因子催化的氧化攻击。提出了一种DAHPS失活机制，其特征是一个金属氧化还原循环，该循环需要其两个活性位点半胱氨酸的顺序氧化。
