Significant enhancement in the binding of p-nitrophenyl-β--xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86 |
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Authors: | Atsushi Kuno Daisuke Shimizu Satoshi Kaneko Tsunemi Hasegawa Yasuo Gama Kiyoshi Hayashi Isao Kusakabe Kazunari Taira |
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Abstract: | 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-nitrophenyl-β--xylobioside substrate was increased by 103-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state. |
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Keywords: | mutant streptomyces site directed mutagenesis xylan endo 1,3 beta xylosidase |
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