Kuroki R, Weaver L H, Matthews B W
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku, Yokohama 236 Japan.
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):8949-54. doi: 10.1073/pnas.96.16.8949.
In contrast to hen egg-white lysozyme, which retains the beta-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 --> His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 --> His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 --> His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 --> His, Asp-20 --> Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the beta-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the alpha-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct.
与在产物中保留底物β构型的鸡蛋清溶菌酶不同，T4溶菌酶（T4L）是一种构型翻转的糖苷酶。然而，将苏氨酸-26替换为组氨酸会使T4L从构型翻转酶转变为构型保留酶。本文表明，苏氨酸-26替换为组氨酸的突变体也是一种转糖苷酶。实际上，转糖基化反应可能比水解反应更有效。相比之下，野生型T4L没有可检测到的转糖苷酶活性。这些结果支持了之前的假设，即苏氨酸-26替换为组氨酸的突变体通过共价中间体进行催化。T26H突变体溶菌酶的进一步突变（谷氨酸-11替换为组氨酸、天冬氨酸-20替换为半胱氨酸）表明，该突变体的催化机制需要谷氨酸-11作为广义酸，但天冬氨酸-20并非必需。这些结果有助于对糖苷酶的活性进行全面的合理化解释，其中底物β侧高度保守的酸性基团（T4L中的谷氨酸-11、鸡蛋清溶菌酶中的谷氨酸-35）作为质子供体，而底物α侧残基的位置和化学特性的改变可导致构型保留或不保留的催化作用、转糖苷酶活性或形成稳定的酶-底物加合物。
