Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic

Abstract

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and ⁴H₃ conformer mimic of a glucoside, binds very tightly to a barley β-D-glucan glucohydrolase, with a Ki constant of 2 x 10⁻⁹ M and a ΔG of 51 kJ mol⁻¹. PheGlcIm binds to the barley β-D-glucan glucohydrolase ~2 x 10⁵ times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10⁶ times tighter than 4-nitrophenyl β-D-glucopyranoside, and 2 x 10⁷ tighter than glucose. The three-dimensional structure of the β-D-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 σlevel form between the three active site residues Asp⁹⁵, His²⁰⁷, and Asp²⁸⁵, and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the β-D-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 Å. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the ⁴E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.