ser - 451和ser - 452参与催化的人类gamma-glutamyl转肽酶。
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Ikeda Y,藤井裕久J,安德森我,谷口N,迈斯特
ser - 451和ser - 452参与催化的人类gamma-glutamyl转肽酶。
生物化学杂志。1995年9月22日;270 (38):22223 - 8。
- PubMed ID
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7673200 (在PubMed]
- 文摘
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所需的丝氨酸残基的催化gamma-glutamyl转肽酶被特定站点的诱变守恒的丝氨酸残基序列比对的基础上的光亚基人,鼠,猪和两个细菌酶。重组人类gamma-glutamyl转肽酶和替换这些丝氨酸残基的阿拉巴马州使用baculovirus-insect细胞表达系统。替换的阿拉巴马州ser - 385, -413年或-425年取得了几乎完全活跃的酶。然而,阿拉巴马州的替换ser - 451或-452产生酶只有1%像野生型酶活性。进一步,他们的双突变体只有0.002%的野生型一样活跃。动能转肽作用使用glycylglycine作为受体分析表明ser - 451和-452突变体的Vmax值大大降低(约3%的野生型);然而,他们的Km值L-gamma-glutamyl-p-nitroanilide捐赠只增加约5比野生型的褶皱。ser - 451和-452的双重突变进一步减少Vmax价值只有0.005%的野生型,虽然这种突变产生影响小(2倍增加)的Km值捐献者。的水解反应的动力学值L-gamma-glutamyl-p-nitroanilide在随后的突变体为转肽作用类似的趋势。ser - 451, -452的失活率和双突变体酶acivicin,强有力的抑制剂,不到1%的野生型酶。 The Ki value of the double mutant for L-serine as a competitive inhibitor of the gamma-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and -452 are located at the position able to interact with the gamma-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.