Clugston S L, Barnard J F, Kinach R, Miedema D, Ruman R, Daub E, Honek J F
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Biochemistry. 1998 Jun 16;37(24):8754-63. doi: 10.1021/bi972791w.
The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.
普遍存在的乙二醛酶系统利用谷胱甘肽（GSH）作为辅因子，将有毒的α-酮醛转化为相应的无毒2-羟基羧酸。该系统中的第一种酶，乙二醛酶I（GlxI），催化GSH与细胞毒性α-酮醛之间非酶促形成的半硫代乙醛的异构化反应。为了研究大肠杆菌的GlxI酶，编码该蛋白的DNA（gloA）被分离出来并整合到质粒pTTQ18中。gloA基因的核苷酸测序预测其编码一个由135个氨基酸组成、分子量为14919的多肽。gloA基因已在大肠杆菌中过表达，并被证明编码GlxI。开发了一种有效的两步纯化方案，每升培养物可产生150 - 200毫克的纯蛋白。电喷雾质谱法证实了纯化蛋白的单体分子量，而凝胶过滤分析表明GlxI是一种30 kDa的同型二聚体。在人类和酿酒酵母的GlxI中发现的天然金属离子锌，对大肠杆菌GlxI的活性没有影响。相反，向生长培养基或纯化的大肠杆菌脱辅基GlxI中添加NiCl₂可大大增强酶活性。电感耦合等离子体和原子吸收分析表明，每个二聚体酶仅结合一个镍离子，这表明该同型二聚体酶中只有一个功能性活性位点。此外，脱辅基蛋白与一摩尔当量的氯化镍反应后恢复了最大活性，这表明金属结合紧密。还研究了pH对镍激活酶动力学的影响。这是首个非锌激活的GlxI的例子，其最大激活是由Ni²⁺实现的。
