Kwon D S, Lin C H, Chen S, Coward J K, Walsh C T, Bollinger J M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Jan 24;272(4):2429-36. doi: 10.1074/jbc.272.4.2429.
The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1, 4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the opposing hydrolysis of this amide bond (Bollinger, J. M., Jr., Kwon, D. S., Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) J. Biol. Chem. 270, 14031-14041). In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains. In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules. A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein. A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs. This observation suggests that the amidase activity in the full-length protein is negatively autoregulated. The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage. No metal ion is required for the amidase activity. A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki approximately 10 microM) but does not inhibit the amidase activity.
来自大肠杆菌的双功能谷胱甘肽亚精胺合成酶/酰胺酶既能催化亚精胺（N-(3-氨基)丙基-1,4-二氨基丁烷）的N1与谷胱甘肽（γ-Glu-Cys-Gly）的甘氨酸羧基之间依赖ATP形成酰胺键，也能催化该酰胺键的反向水解反应（博林格，J.M.，小权，D.S.，惠斯曼，G.W.，科尔特，R.，以及沃尔什，C.T.（1995年）《生物化学杂志》270，14031 - 14041）。在我们之前描述其初步特性的工作中，我们提出这个619个氨基酸（70 kDa）的蛋白质可能具有独立折叠且有功能的酰胺酶（N端）和合成酶（C端）结构域。在本研究中，我们通过表达独立折叠且有功能的酰胺酶和合成酶模块证实了这一假设。一个包含C端431个氨基酸（50 kDa）的片段仅具有合成酶活性，其稳态动力学参数与全长蛋白相似。一个包含N端225个氨基酸（25 kDa）的片段仅具有酰胺酶活性，并且相对于全长蛋白，它对谷胱甘肽亚精胺类似物的水解有显著激活作用。这一观察结果表明全长蛋白中的酰胺酶活性存在负向自动调节。酰胺酶活性位点催化谷胱甘肽的酰胺和酯衍生物（如谷胱甘肽乙酯和谷胱甘肽酰胺）的水解，但对乙酰亚精胺（N1和N8）和乙酰精胺（N1）无活性，这表明谷胱甘肽为谷胱甘肽亚精胺酰胺键的裂解提供了主要的识别决定因素。酰胺酶活性不需要金属离子。一种设计为模拟任一反应中假定的四面体中间体的谷胱甘肽亚精胺的四面体膦酸酯类似物，可抑制合成酶活性（Ki约为10 μM），但不抑制酰胺酶活性。
