Bollinger J M, Kwon D S, Huisman G W, Kolter R, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1995 Jun 9;270(23):14031-41. doi: 10.1074/jbc.270.23.14031.
Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W. (1975) J. Biol. Chem. 250, 2648-2654). Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.
锥虫科和大肠杆菌的谷胱甘肽亚精胺（GSP）合成酶将ATP水解（生成ADP和磷酸）与亚精胺（N-（3-氨丙基）-1,4-二氨基丁烷）和谷胱甘肽的甘氨酸羧基（γ-Glu-Cys-Gly）之间酰胺键的形成偶联起来。在致病性锥虫中，该反应是抗氧化代谢物三胺（N1,N8-双（谷胱甘肽基）亚精胺）生物合成的倒数第二步，是药物设计的靶点。在本研究中，从大肠杆菌B中纯化GSP合成酶至接近均一，分离并测序了编码它的基因，大量过量表达并纯化了该酶，并对重组酶进行了表征。发现该70 kDa蛋白具有意想不到的第二种催化活性，即谷胱甘肽亚精胺酰胺键水解。因此，双功能GSP合成酶/酰胺酶催化相反的酰胺键形成和裂解反应，并伴有ATP的净水解。合成酶活性通过从C末端进行81个残基的蛋白水解切割而被选择性废除，这表明这两种活性存在于不同的结构域（N末端酰胺酶和C末端合成酶）。在没有底物的情况下，该位点的蛋白水解很容易进行，但在ATP、谷胱甘肽和Mg2+存在时受到抑制。使用一系列类似物来探测合成酶活性的亚精胺结合位点。二氨基丙烷作为底物的活性、C4-C8二氨基烷烃的无活性以及3-氨丙基部分修饰的类似物比4-氨基丁基部分修饰的类似物特异性丧失更大，表明该酶主要识别亚精胺的二氨基丙烷部分，并证实N-1（氨丙基N）为谷胱甘肽连接位点（Tabor，H.和Tabor，C.W.（1975年）《生物化学杂志》250，2648-2654）。一组二氟取代亚精胺衍生物的Km和kcat趋势表明，该酶可能结合胺亲核试剂的次要去质子化形式。
