PDBsum entry 1qt3

Hydrolase

PDB id

1qt3

Contents

			Protein chain

					164 a.a. *

			Ligands

					HED

			Metals

					_CL ×2

			Waters ×159

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis of the conversion of t4 lysozyme into a transglycosidase by reengineering the active site.

Authors

R.Kuroki, L.H.Weaver, B.W.Matthews.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 8949-8954. [DOI no: 10.1073/pnas.96.16.8949]

PubMed id

10430876

Abstract

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.



	Figure 2. Fig. 2. Comparison of the products formed by the digestion of the substrate shown in Fig. 1 with WT lysozyme and mutant T26H. (a) Substrate alone. (b) Digestion of 2 × 10^ 6 M substrate by 8 × 10^ 8 M WT* lysozyme for 5 min at 25°C (14). (c) Digestion of 2 × 10^ 6 M substrate by 5 × 10^ 7 M T26H for 5 min at 25°C (14). (d) Digestion of 8 × 10^ 5 M substrate by 5 × 10^ 7 M T26H for 60 min at 0°C. See text for additional details and explanation of peaks.		Figure 3. Fig. 3. Superposition of 12 structures of mutant lysozymes (see text). The figure includes WT*, E11H, E11N, D20A, D20S, D20N, D20C, D20E, T26D, T26H, T26Q, and T26E with the covalent adduct formed by this mutant (13). Water molecules clustered together at the two sites discussed in the text are shown as red spheres. The water molecule present in the covalent adduct also is shown as a red sphere. Other solvent molecules in the vicinity of the active site are shown as red crosses.