Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Nov;5(11):2319–2328. doi: 10.1002/pro.5560051118

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

L A Plesniak 1, G P Connelly 1, W W Wakarchuk 1, L P McIntosh 1
PMCID: PMC2143293  PMID: 8931150

Abstract

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 1H, 13C, and 15N 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 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees 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.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson D. E., Becktel W. J., Dahlquist F. W. pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. Biochemistry. 1990 Mar 6;29(9):2403–2408. doi: 10.1021/bi00461a025. [DOI] [PubMed] [Google Scholar]
  2. Bachovchin W. W. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry. 1986 Nov 18;25(23):7751–7759. doi: 10.1021/bi00371a070. [DOI] [PubMed] [Google Scholar]
  3. Blomberg F., Maurer W., Rüterjans H. Nuclear magnetic resonance investigation of 15N-labeled histidine in aqueous solution. J Am Chem Soc. 1977 Dec 7;99(25):8149–8159. doi: 10.1021/ja00467a005. [DOI] [PubMed] [Google Scholar]
  4. Burley S. K., Petsko G. A. Weakly polar interactions in proteins. Adv Protein Chem. 1988;39:125–189. doi: 10.1016/s0065-3233(08)60376-9. [DOI] [PubMed] [Google Scholar]
  5. Coughlan M. P., Hazlewood G. P. beta-1,4-D-xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol Appl Biochem. 1993 Jun;17(Pt 3):259–289. [PubMed] [Google Scholar]
  6. Davies G., Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure. 1995 Sep 15;3(9):853–859. doi: 10.1016/S0969-2126(01)00220-9. [DOI] [PubMed] [Google Scholar]
  7. Davoodi J., Wakarchuk W. W., Campbell R. L., Carey P. R., Surewicz W. K. Abnormally high pKa of an active-site glutamic acid residue in Bacillus circulans xylanase. The role of electrostatic interactions. Eur J Biochem. 1995 Sep 15;232(3):839–843. [PubMed] [Google Scholar]
  8. Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
  9. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Griffin J. H., Cohen J. S., Schechter A. N. The assignment of an exchangeable low-field NH proton resonance of ribonuclease A to the active-site histidine-119. Biochemistry. 1973 May 22;12(11):2096–2099. doi: 10.1021/bi00735a012. [DOI] [PubMed] [Google Scholar]
  11. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lawson S. L., Wakarchuk W. W., Withers S. G. Effects of both shortening and lengthening the active site nucleophile of Bacillus circulans xylanase on catalytic activity. Biochemistry. 1996 Aug 6;35(31):10110–10118. doi: 10.1021/bi960586v. [DOI] [PubMed] [Google Scholar]
  13. Liang T. C., Abeles R. H. Complex of alpha-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy. Biochemistry. 1987 Dec 1;26(24):7603–7608. doi: 10.1021/bi00398a011. [DOI] [PubMed] [Google Scholar]
  14. Markley J. L. Hydrogen bonds in serine proteinases and their complexes with protein proteinase inhibitors. Proton nuclear magnetic resonance studies. Biochemistry. 1978 Oct 31;17(22):4648–4656. doi: 10.1021/bi00615a010. [DOI] [PubMed] [Google Scholar]
  15. McIntosh L. P., Hand G., Johnson P. E., Joshi M. D., Körner M., Plesniak L. A., Ziser L., Wakarchuk W. W., Withers S. G. The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase. Biochemistry. 1996 Aug 6;35(31):9958–9966. doi: 10.1021/bi9613234. [DOI] [PubMed] [Google Scholar]
  16. Miao S., Ziser L., Aebersold R., Withers S. G. Identification of glutamic acid 78 as the active site nucleophile in Bacillus subtilis xylanase using electrospray tandem mass spectrometry. Biochemistry. 1994 Jun 14;33(23):7027–7032. doi: 10.1021/bi00189a002. [DOI] [PubMed] [Google Scholar]
  17. Pelton J. G., Torchia D. A., Meadow N. D., Roseman S. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques. Protein Sci. 1993 Apr;2(4):543–558. doi: 10.1002/pro.5560020406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Plesniak L. A., Wakarchuk W. W., McIntosh L. P. Secondary structure and NMR assignments of Bacillus circulans xylanase. Protein Sci. 1996 Jun;5(6):1118–1135. doi: 10.1002/pro.5560050614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Richardson J. S., Richardson D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science. 1988 Jun 17;240(4859):1648–1652. doi: 10.1126/science.3381086. [DOI] [PubMed] [Google Scholar]
  20. Sancho J., Serrano L., Fersht A. R. Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability. Biochemistry. 1992 Mar 3;31(8):2253–2258. doi: 10.1021/bi00123a006. [DOI] [PubMed] [Google Scholar]
  21. Sheard B., Yamane T., Shulman R. G. Nuclear magnetic resonance study of cyanoferrimyoglobin; identification of pseudocontact shifts. J Mol Biol. 1970 Oct 14;53(1):35–48. doi: 10.1016/0022-2836(70)90044-6. [DOI] [PubMed] [Google Scholar]
  22. Stoesz J. D., Malinowski D. P., Redfield A. G. Nuclear magnetic resonance study of solvent exchange and nuclear Overhauser effect of the histidine protons of bovine superoxide dismutase. Biochemistry. 1979 Oct 16;18(21):4669–4675. doi: 10.1021/bi00588a030. [DOI] [PubMed] [Google Scholar]
  23. Tanokura M. 1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides. Biochim Biophys Acta. 1983 Feb 15;742(3):576–585. doi: 10.1016/0167-4838(83)90276-5. [DOI] [PubMed] [Google Scholar]
  24. Törrönen A., Harkki A., Rouvinen J. Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site. EMBO J. 1994 Jun 1;13(11):2493–2501. doi: 10.1002/j.1460-2075.1994.tb06536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Van Dijk A. A., Scheek R. M., Dijkstra K., Wolters G. K., Robillard G. T. Characterization of the protonation and hydrogen bonding state of the histidine residues in IIAmtl, a domain of the phosphoenolpyruvate-dependent mannitol-specific transport protein. Biochemistry. 1992 Sep 22;31(37):9063–9072. doi: 10.1021/bi00152a050. [DOI] [PubMed] [Google Scholar]
  26. Wakarchuk W. W., Campbell R. L., Sung W. L., Davoodi J., Yaguchi M. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 1994 Mar;3(3):467–475. doi: 10.1002/pro.5560030312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wishart D. S., Bigam C. G., Yao J., Abildgaard F., Dyson H. J., Oldfield E., Markley J. L., Sykes B. D. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR. 1995 Sep;6(2):135–140. doi: 10.1007/BF00211777. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES