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Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: Nmr assignments, pH titration, and hydrogen exchange
Author(s) -
Plesniak Leigh A.,
Connelly Gregory P.,
Mcintosh Lawrence P.,
Wakarchuk Warren W.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560051118
Subject(s) - chemistry , bacillus circulans , tautomer , histidine , heteronuclear molecule , hydrogen bond , protonation , imidazole , nuclear magnetic resonance spectroscopy , crystallography , solvent , titration , molecule , stereochemistry , organic chemistry , enzyme , ion
Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic‐aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1 H, 13 C, and 15 N resonances and tautomeric forms of the imidazole rings were obtained from two‐dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1 H ϕ2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 × 10 −5 s −1 at pH * 7.04 and 30°C) is retarded by > 10 6 ‐fold relative to an exposed histidine. The p K a of His 156 is unperturbed at ˜6.5, as measured from the pH dependence of the 15 N‐ and 1 H‐NMR spectra of BCX. In contrast, His 149 has a p K a < 2.3, existing in the neutral N ϕ2 H tautomeric state under all conditions examined. BCX unfolds at low pH and 30°C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.