期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：16
页码：8949-8954
DOI：10.1073/pnas.96.16.8949
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 -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.
