Significant enhancement in the binding of p-nitrophenyl-beta-D-xylobiosideby the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F /10 xylanase by a His residue. To our surprise, the affinity for the p-nitr ophenyl-beta-D-xylobioside substrate was increased by 10(3)-fold in the cas e of the mutant E128H enzyme compared with that of the wild-type F/10 xylan ase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native x ylanase and a nitrogen in the mutant) and the alpha-carbon was barely chang ed. Thus, the alteration of the acid/base functionality (Glu-128 to His mut ation) provided a significantly favorable interaction within the E128H enzy me/substrate complex in the ground state, accompanying, a reduction in the stabilization effect in the transition state, (C) 1999 Federation of Europe an Biochemical Societies.