PKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

Shuguang Yuan,Wim Van Den Ende,Willem Lammens,Katrien Le Roy,Marc De Maeyer,Tom Venken,Beata G Vertessy

doi:10.1371/journal.pone.0037453

Abstract

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst.

Highlights

Carbohydrates play an important role in a diverse array of biological processes
We checked the predictive power of sucrose docking simulations in this system and confirmed that Glide [31,32] in Schrodinger 2011 could position sucrose in a correct way in the active site of AtcwINV1, as observed in the mutated crystal structures 2QQU, 2QQV and 2QQW (Figures 2 & 3)
The substrate binding mode in AtcwINV1 In the docked AtcwINV1 (2AC1)/sucrose complex, the sucrose molecule is stabilized by a series of H-bonds including a 2.8 A Hbond between the glycosidic oxygen O1 and acid/base E203, a distance allowing proton transfer from E203 to the glycosidic oxygen

Summary

Introduction

Carbohydrates play an important role in a diverse array of biological processes. Their functional and structural variety implies a large amount of enzymes involved in their modification, synthesis and breakdown. Glycoside hydrolases (GH) split the glycosidic bond between two carbohydrates or between a carbohydrate and an aglycon moiety. They play important roles both in plants and micro-organisms. Naumoff proposed that clan GH-J should be combined with a-arabinases and b-xylosidases [5] (families GH43 and GH62) into one ‘‘b-fructosidase superfamily’’

Methods

Results

Conclusion