Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Apr 1;371(Pt 1):175–181. doi: 10.1042/BJ20021447

pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase.

Yoko Kato-Toma 1, Takashi Iwashita 1, Katsuyoshi Masuda 1, Yoshiaki Oyama 1, Masaji Ishiguro 1
PMCID: PMC1223266  PMID: 12513696

Abstract

13C-NMR spectroscopy was used to estimate the p K a values for the Tyr(150) (Y150) residue in wild-type and mutant class C beta-lactamases. The tyrosine residues of the wild-type and mutant lactamases were replaced with (13)C-labelled L-tyrosine ([ phenol -4-(13)C]tyrosine) in order to observe the tyrosine residues selectively. Spectra of the wild-type and K67C mutant (Lys(67)-->Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr(150). Titration experiments showed that the chemical shift of the Tyr(150) resonance in the wild-type enzyme is almost invariant in a range of 0.1 p.p.m. up to pH 11 and showed that the p K (a) of this residue is well above 11 in the substrate-free form. According to solvent accessibility calculations on X-ray-derived structures, the phenolic oxygen of Tyr(150), which is near the amino groups of Lys(315) and Lys(67), appears to have low solvent accessibility. These results suggest that, in the native enzyme, Tyr(150) in class C beta-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr(150) loses a proton, a proton from Lys(67) would replace it. Consequently, Tyr(150) would be protonated during the entire titration.

Full Text

The Full Text of this article is available as a PDF (152.6 KB).

Selected References

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

  1. Adediran S. A., Pratt R. F. Beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: comparisons with a structurally similar class C beta-lactamase. Biochemistry. 1999 Feb 2;38(5):1469–1477. doi: 10.1021/bi982308x. [DOI] [PubMed] [Google Scholar]
  2. Beadle Beth M., Trehan Indi, Focia Pamela J., Shoichet Brian K. Structural milestones in the reaction pathway of an amide hydrolase: substrate, acyl, and product complexes of cephalothin with AmpC beta-lactamase. Structure. 2002 Mar;10(3):413–424. doi: 10.1016/s0969-2126(02)00725-6. [DOI] [PubMed] [Google Scholar]
  3. Caselli E., Powers R. A., Blasczcak L. C., Wu C. Y., Prati F., Shoichet B. K. Energetic, structural, and antimicrobial analyses of beta-lactam side chain recognition by beta-lactamases. Chem Biol. 2001 Jan;8(1):17–31. doi: 10.1016/s1074-5521(00)00052-1. [DOI] [PubMed] [Google Scholar]
  4. Crichlow G. V., Kuzin A. P., Nukaga M., Mayama K., Sawai T., Knox J. R. Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion. Biochemistry. 1999 Aug 10;38(32):10256–10261. doi: 10.1021/bi9908787. [DOI] [PubMed] [Google Scholar]
  5. Damblon C., Raquet X., Lian L. Y., Lamotte-Brasseur J., Fonze E., Charlier P., Roberts G. C., Frère J. M. The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1747–1752. doi: 10.1073/pnas.93.5.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dubus A., Ledent P., Lamotte-Brasseur J., Frère J. M. The roles of residues Tyr150, Glu272, and His314 in class C beta-lactamases. Proteins. 1996 Aug;25(4):473–485. doi: 10.1002/prot.7. [DOI] [PubMed] [Google Scholar]
  7. Dubus A., Normark S., Kania M., Page M. G. The role of tyrosine 150 in catalysis of beta-lactam hydrolysis by AmpC beta-lactamase from Escherichia coli investigated by site-directed mutagenesis. Biochemistry. 1994 Jul 19;33(28):8577–8586. doi: 10.1021/bi00194a024. [DOI] [PubMed] [Google Scholar]
  8. Dubus A., Wilkin J. M., Raquet X., Normark S., Frère J. M. Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad. Biochem J. 1994 Jul 15;301(Pt 2):485–494. doi: 10.1042/bj3010485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dyson H. J., Jeng M. F., Tennant L. L., Slaby I., Lindell M., Cui D. S., Kuprin S., Holmgren A. Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57. Biochemistry. 1997 Mar 4;36(9):2622–2636. doi: 10.1021/bi961801a. [DOI] [PubMed] [Google Scholar]
  10. Ishiguro M., Imajo S. Modeling study on a hydrolytic mechanism of class A beta-lactamases. J Med Chem. 1996 May 24;39(11):2207–2218. doi: 10.1021/jm9506027. [DOI] [PubMed] [Google Scholar]
  11. Kato-Toma Y., Ishiguro M. Reaction of Lys-Tyr-Lys triad mimics with benzylpenicillin: insight into the role of Tyr150 in class C beta-lactamase. Bioorg Med Chem Lett. 2001 May 7;11(9):1161–1164. doi: 10.1016/s0960-894x(01)00168-8. [DOI] [PubMed] [Google Scholar]
  12. Khare D., Alexander P., Antosiewicz J., Bryan P., Gilson M., Orban J. pKa measurements from nuclear magnetic resonance for the B1 and B2 immunoglobulin G-binding domains of protein G: comparison with calculated values for nuclear magnetic resonance and X-ray structures. Biochemistry. 1997 Mar 25;36(12):3580–3589. doi: 10.1021/bi9630927. [DOI] [PubMed] [Google Scholar]
  13. Lamotte-Brasseur J., Dubus A., Wade R. C. pK(a) calculations for class C beta-lactamases: the role of Tyr-150. Proteins. 2000 Jul 1;40(1):23–28. doi: 10.1002/(sici)1097-0134(20000701)40:1<23::aid-prot40>3.0.co;2-7. [DOI] [PubMed] [Google Scholar]
  14. Lobkovsky E., Billings E. M., Moews P. C., Rahil J., Pratt R. F., Knox J. R. Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a beta-lactamase transition-state analog. Biochemistry. 1994 Jun 7;33(22):6762–6772. doi: 10.1021/bi00188a004. [DOI] [PubMed] [Google Scholar]
  15. Lobkovsky E., Moews P. C., Liu H., Zhao H., Frere J. M., Knox J. R. Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11257–11261. doi: 10.1073/pnas.90.23.11257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Monnaie D., Dubus A., Cooke D., Marchand-Brynaert J., Normark S., Frère J. M. Role of residue Lys315 in the mechanism of action of the Enterobacter cloacae 908R beta-lactamase. Biochemistry. 1994 May 3;33(17):5193–5201. doi: 10.1021/bi00183a024. [DOI] [PubMed] [Google Scholar]
  17. Monnaie D., Dubus A., Frère J. M. The role of lysine-67 in a class C beta-lactamase is mainly electrostatic. Biochem J. 1994 Aug 15;302(Pt 1):1–4. doi: 10.1042/bj3020001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oefner C., D'Arcy A., Daly J. J., Gubernator K., Charnas R. L., Heinze I., Hubschwerlen C., Winkler F. K. Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature. 1990 Jan 18;343(6255):284–288. doi: 10.1038/343284a0. [DOI] [PubMed] [Google Scholar]
  19. Richards F. M. Areas, volumes, packing and protein structure. Annu Rev Biophys Bioeng. 1977;6:151–176. doi: 10.1146/annurev.bb.06.060177.001055. [DOI] [PubMed] [Google Scholar]
  20. Tsukamoto K., Nishida N., Tsuruoka M., Sawai T. Function of the conserved triad residues in the class C beta-lactamase from Citrobacter freundii GN346. FEBS Lett. 1990 Oct 1;271(1-2):243–246. doi: 10.1016/0014-5793(90)80416-g. [DOI] [PubMed] [Google Scholar]
  21. Tsukamoto K., Tachibana K., Yamazaki N., Ishii Y., Ujiie K., Nishida N., Sawai T. Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346. Eur J Biochem. 1990 Feb 22;188(1):15–22. doi: 10.1111/j.1432-1033.1990.tb15365.x. [DOI] [PubMed] [Google Scholar]
  22. Usher K. C., Blaszczak L. C., Weston G. S., Shoichet B. K., Remington S. J. Three-dimensional structure of AmpC beta-lactamase from Escherichia coli bound to a transition-state analogue: possible implications for the oxyanion hypothesis and for inhibitor design. Biochemistry. 1998 Nov 17;37(46):16082–16092. doi: 10.1021/bi981210f. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES