CHEMICAL STUDIES OF Y-GLUTAMYLTRANSFERASE ACTIVE SITE

在我们最近的工作中，我们已经证明了伽玛-谷氨酰转移酶(GGT)，一种 膜结合的酶，完全和不可逆转地失活 硫代巴比妥酸和苯巴比妥。这两种化合物都被证明是 活性部位导向的GGT抑制剂。此外，数据的稳定性 严格变性分离的[~3H]-苯巴比妥-GGT络合物 条件提供了令人信服的证据苯巴比妥是共价的 绑定到GGT。到目前为止，人们对活动站点知之甚少。 GGT中的残留物(S)是催化过程中必不可少的。这个 本研究提案的目的是启动将扩展的研究 并放大我们最初的观察结果，以进一步了解 酶催化所必需的活性中心残基的性质。为 拟议的研究，一大批来自大鼠肾脏的GGT将是 纯净的。新近发现的其他细菌对GGT失活的研究 GGT抑制剂，即四氧嘧啶，4，6-二羟基-2-甲硫基 将继续使用嘧啶、2，4-二羟基-5-硫代嘧啶和尿嘧啶。 与之反应的活性部位氨基酸残基(S)的鉴定 一些精选的嘧啶类抑制剂(如苯巴比妥和 2-S-甲硫代巴比妥酸)和其他已知的不可逆灭活剂 GGT如6-重氮-5-氧代-L-去亮氨酸(DON)和(α， 5S)-Alpha-amino-3-chloro-4，5-dihydro-5-isoxazoleacetic酸(AT-125)将 被执行。在这些研究中，纯化的GGT将用 过量的放射性标记抑制物可使酶完全失活 活动。对于化学修饰残留物的鉴定， 分离标记的GGT小亚基将受到控制 使用选择性和特定的蛋白水解酶进行蛋白水解酶(S)。这个 放射性标记多肽(S)将使用高效等技术进行纯化 高效液相色谱，高压电泳法， 离子交换层析和凝胶过滤层析。纯化后的序列 放射性标记的多肽(S)将使用自动EDMAN进行测定 降解法。随后，化学修饰残留物(S)将 用氨基酸分析仪进行鉴定。这些研究对于 了解GGT中活性中心残留物(S)对 并了解GGT的作用机制。另外， 这些研究可能有助于开发特异性的GGT不可逆抑制剂。 它们在活体内很活跃。这反过来可能有助于理解 GGT的生理作用。

In our recent work, we have shown that Gamma-glutamyltransferase (GGT), a membrane-bound enzyme, is completely and irreversibly inactivated by thiobarbituric acid and phenobarbital. Both the compounds were shown to be active-site directed inhibitors of GGT. Also, the stability of the isolated [3H]-phenobarbital-GGT complex under rigorous denaturation conditions provided compelling evidence that phenobarbital is covalently bound to GGT. Little is known, to date, regarding the active-site residue(s) in GGT that are essential for the catalytic process. The purpose of this research proposal is to initiate studies which will extend and amplify our initital observations to gain further insights into the nature of the active-site residues essential for enzymic catalysis. For the proposed studies, a large batch of GGT from rat kidneys will be purified. Studies on the inactivation of GGT by other recently discovered inhibitors of GGT, i.e., alloxan, 4,6-dihydroxy-2-methylmercapto pyrimidine, 2,4-dihydroxy-5-thio-pyrimidine and uracil will be continued. Identification of active-site amino acid residue(s) that are reactive to some selected pyrimidine inhibitors (e.g., phenobarbital and 2-S-methylthiobarbituric acid) and other known irreversible inactivators of GGT such as 6-diazo-5-oxo-L-norleucine (DON) and (AlphaS, 5S)-Alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) will be carried out. For these studies, the purified GGT will be treated with an excess of radiolabeled-inhibitor for complete inactivation of the enzyme activity. For identification of chemically-modified residues, the separated labeled small subunit of GGT will be subjected to controlled proteolysis using selective and specific proteolytic enzyme(s). The radiolabeled peptide(s) will be purified using techniques such as high performance liquid chromatography, high voltage electrophoresis, ion-exchange and gel filtration chromatography. The sequence of purified radiolabeled peptides(s) will be determined using automated Edman degradation method. Subsequently, the chemically-modified residue(s) will be identified using an amino-acid analyzer. These studies are essential to understand the active-site residue(s) in GGT that are responsbile for the catalytic process and to understand the mechanism of GGT action. Also, these studies may aid in developing specific irreversible inhibitors of GGT that are active in vivo. This, in turn, may help understand the physiological role of GGT.