R. Kuroki et al., Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site, P NAS US, 96(16), 1999, pp. 8949-8954
Citations number
28
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
In contrast to hen egg-white lysozyme, which retains the beta-configuration
of the substrate in the product, T4 lysozyme (T4L) is an inverting glgcosi
dase. The substitution Thr-26 --> His, however, converts T4L from an invert
ing 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 detectabl
e 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 lys
ozyme 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 configura
tion, to transglycosidase activity, or to the formation of a stable enzyme-
substrate adduct.