Structural basis for the inhibition of 1,3-1,4-β-D-glucanase by noncompetitive calcium ion and competitive Tris inhibitors

Abstract

In this paper, we determine the mutant W203F structure of TFsβ-glucanase, which contains aromatic residue Trp203 at the active site of the enzyme. Residue Trp203 is stacked with the glucose product of cellotriose. Further analysis reveals that two extra calcium ions and a Tris molecule bind to the mutant structure. A Tris molecule, bound to the catalytic residues of Glu56 and Glu60, was found at the position normally taken by substrate binding at the -1 subsite. In addition, a second Ca(2+) ion was found near the residues Phe152 and Glu154 on the protein's surface, and a third one near the active site residue Asp202. Kinetic experiments reveal that both Tris and imidazole are competitive inhibitors, while calcium is a noncompetitive inhibitor for TFsβ-glucanase. The two types of enzymatic inhibition are first-time descriptions for the glycosyl hydrolase family 16.