title: The role of conserved arginine residue in loop 4 of glycoside hydrolase family 10 xylanases.
abstract: An arginine residue in loop 4 connecting beta strand 4 and alpha-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K(m) and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.
authors.affiliation: National Food Research Institute, Ibaraki, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Nishimoto
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Hayashi
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology; D017124:Conserved Sequence; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D001120:Arginine; D043364:Endo-1,4-beta Xylanases