ABNORMALLY HIGH PK(A) OF AN ACTIVE-SITE GLUTAMIC-ACID RESIDUE IN BACILLUS-CIRCULANS XYLANASE - THE ROLE OF ELECTROSTATIC INTERACTIONS

The active site of Bacillus circulans xylanase (1,4-beta-D-xylanohydro lase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu17 2, which are crucial for the catalytic activity of the enzyme. Fourier -transform infrared spectroscopy was used to determine the ionization state of these residues as a function of pH. For the wild-type enzyme, titration of one of the carboxylate groups occurs at pH 6.8. This tit ration is absent in the Glu78-->Gln and Glu172-->Gln variants of the e nzyme. This, together with crystallographic data, indicates that Glu17 2 has an abnormally high pK(a) of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential sc anning calorimetry experiments with the wild-type xylanase and a numbe r of its mutants have shown that the presence of two nearby carboxyl g roups results in a pH-dependent destabilization of the protein structu re.