ASPARAGINE-127 OF XYLANASE-A FROM STREPTOMYCES-LIVIDANS, A KEY RESIDUE IN GLYCOSYL HYDROLASES OF SUPERFAMILY-4 7 - KINETIC EVIDENCE FOR ITSINVOLVEMENT IN STABILIZATION OF THE CATALYTIC INTERMEDIATE/

Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA ) from Streptomyces lividans, belonging to family 10 and superfamily 4 /7 of glycosyl hydrolases, was chosen to study the role of this conser ved residue. The isosteric mutation N127D introduced did not affect th e fold of XlnA as revealed by circular dichroism. Comparison of the ki netic constants of N127D and wild-type XlnA revealed a 70-fold decreas e in the specificity constant (k(cat)/K-M) towards birchwood xylan, wh ich is attributed solely to the difference in the k(cat) value and ind icates a role of N127 in stabilization of the catalytic intermediate. N127 also plays a role in maintaining the ionization states of the two catalytic residues, as shown by the modified pH profile of XlnA-N127D . Characterization of XlnA-N127D and the analysis of the three-dimensi onal structure of XlnA converge toward a stabilization role for N127 i n the catalytic site of XlnA.