Recognition of reactive high-energy conformations by shape complementarity and specific enzyme–substrate interactions in family 10 and 11 xylanases

Abstract

Binding of xylotetraose to the family 10 xylanase (CEX) from Cellulomonas fimi and the family 11 xylanase (BCX) from Bacillus circulans was investigated using quantum mechanical calculations, molecular dynamics simulations and molecular mechanics–Poisson–Boltzmann surface area (MM-PBSA) free energy calculations. In the reactive enzyme–substrate conformation, in which the catalytic groups are precisely positioned for the catalytic reaction, the −1 sugar moiety of the substrate adopted a B_3,O (boat) conformation for CEX and a ²S_O (skew-boat) conformation for BCX. Upon binding the internal energies of the reactive substrate conformations are increased by about 9 kJ mol⁻¹. In contrast to this, the internal energies of substrates in all-chair conformation are increased by 33–43 kJ mol⁻¹, with somewhat larger increase for BCX than CEX. As a consequence, the stability order of the all-chair conformation, which is the most stable conformation in aqueous solution, and the distorted reactive conformations is reversed upon binding to the enzyme. Thus, in addition to specific enzyme–substrate interactions, CEX and especially BCX recognise the reactive high-energy conformation by the substrate-binding site of several sugar-binding subsites complementary in shape to the reactive conformation. The role of substrate distortion in glycosidase-catalysed reactions is discussed.