Abnormally High pK_a of an Active‐Site Glutamic Acid Residue in Bacillus Circulans Xylanase

Abstract

The active site of Bacillus circulans xylanase (1,4‐β‐D‐xylanohydrolase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu172, 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 titration is absent in the Glu78→Gln and Glu172→Gln variants of the enzyme. This, together with crystallographic data, indicates that Glu172 has an abnormally high pKa of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential scanning calorimetry experiments with the wild‐type xylanase and a number of its mutants have shown that the presence of two nearby carboxyl groups results in a pH‐dependent destabilization of the protein structure.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

“…The apparent pK a values for Glu177 and Glu86 in XynII have been estimated to be around 7 and 4 on the basis of the pH dependence of activity (37). The pK a values of corresponding Glu residues in B. circulans xylanase (BCX) have been determined using infrared spectroscopy and 13 C NMR spectroscopy to be very similar to those in XynII, at 6.7 and 4.6, respectively (33,38,39). The active site residues are conserved in both enzymes; therefore, similar pK a values are expected.…”

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family

“…However, as suggested by White and Rose (1997), in all b-glycosyl hydrolases the pK a of the acidic residue Glu-207 in SG is expected to undergo significant changes of two to three pH units during the reaction. The high pK a of the proton donor Glu-207 might be due to electrostatic interactions with other carboxylate groups, as is the case for xylanases (Davoodi et al, 1995), or might be dependent upon a local pronounced hydrophobic environment (Keresztessy et al, 1994). Inspection of the binding pocket of SG around Glu-207 and Glu-416 indicates that both catalytic amino acids are shielded by a number of hydrophilic residues, which all are within a distance of 3.6 to 4.9 Å .…”

Complete measurement of the pKavalues of the carboxyl and imidazole groups inBacillus circulansxylanase

“…Indeed, because the stability of BCX decreases with pH such that this protein unfolds readily under strongly acidic conditions (Davoodi et al, 1995;Plesniak et al, 1996a), we conclude that these two aspartate groups are always ionized within the context of the native enzyme. Based on the crystal structure of BCX (Campbell et al, 1993), Asp 83 is buried entirely in the interior of the protein, forming hydrogen bonds with the positively charged guanido group of Arg 136 and the phenolic 0% of Tyr 53 and Tyr 105 (Fig.…”