A stable isotope-aided NMR study of the active site of an endoglucanase from a strain of Bacillus.

摘要: Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression activity. Endoglucanase K, was specifically labeled with [indole-2-13C]Trp, prepared recombinant subtilis that carried gene this enzyme on an vector, pHSP-KC331. Twelve cross-peaks originating C-2 position residues were separately observed 1H-13C heteronuclear spectrum, and six have assigned site-specifically by using site-directed mutagenesis. The chemical shifts Trp-174 Trp-243 affected addition cellotriose as a competitive inhibitor enzyme. On basis data obtained after modification N-bromosuccinimide, it appears oxidized first retention 56% original activity then complete loss Substitution or Tyr residue caused decrease specific 49 8% wild-type enzyme, respectively. Km values these mutant enzymes p-nitrophenyl beta-D-cellotrioside increased 5 8 times those respectively, while kcat both decreased one-fifth enzymes. These results suggest play role binding substrate and/or catalytic