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

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