The Role of Conserved Arginine Residue in Loop 4 of Glycoside Hydrolase Family 10 Xylanases

抄録

An arginine residue in loop 4 connecting β strand 4 and α-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg²⁰⁴ in xylanase A from Bacillus halodurans C-125 (XynA) and Arg¹⁹⁶ 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.