Abstract
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-kcat⁄Km and the pH-kcat relationships of these mutant enzymes were measured. The pKe2 and pKes2 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 pKes2 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 pKa 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.
