Crystal structures of glutathione- and inhibitor-bound human GGT1: critical interactions within the cysteinylglycine binding site.
Crystal structures of glutathione- and inhibitor-bound human GGT1: critical interactions within the cysteinylglycine binding site.
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DOI：
10.1074/jbc.ra120.016265
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Hanigan MH
中科院分区：
文献类型：
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作者：
Terzyan SS;Nguyen LT;Burgett AWG;Heroux A;Smith CA;You Y;Hanigan MH
γ-谷氨酰转肽酶（GGT 1）的过度表达与一系列人类疾病有关，包括哮喘、再灌注损伤和癌症。抑制剂是治疗所必需的，但是由于缺乏关于底物结合和裂解的结构信息，GGT 1的有效特异性抑制剂的开发受到阻碍。为了提高我们对底物裂解的分子机制的理解，我们已经解决了谷胱甘肽（底物）和磷酸-谷胱甘肽类似物（一种不可逆的抑制剂）结合在活性位点的人GGT 1（hGGT 1）的晶体结构。这些是第一个结构的任何真核GGT与半胱氨酰甘氨酸区的底物结合位点被占领。这些结构和apo-hGGT的结构揭示了当底物结合时活性位点内氨基酸残基的移动。Asn-401和Thr-381各自与跨越γ-谷氨酰键的两个GSH原子形成氢键。hGGT 1的三个不同原子与GSH的半胱氨酸的羧基氧相互作用。酶和底物之间的相互作用随着底物向活性位点裂缝的更深处移动而改变。基片重新取向，在基片和含氧阴离子空穴之间形成新的氢键。当底物和酶之间形成酰基键时，Thr-381被锁定为单一构象。这些数据提供了深入的hGGT 1的底物特异性的分子水平上，并提供了一个看似不同的观察hGGT 1突变体的酶活性的解释。这些知识将有助于设计临床上有用的hGGT 1抑制剂。
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.