Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jul 1;365(Pt 1):303–309. doi: 10.1042/BJ20011468

Role of alphaArg145 and betaArg263 in the active site of penicillin acylase of Escherichia coli.

Wynand B L Alkema 1, Antoon K Prins 1, Erik de Vries 1, Dick B Janssen 1
PMCID: PMC1222674  PMID: 12071857

Abstract

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, alphaArg145 and betaArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants alphaArg145-->Leu (alphaArg145Leu), alphaArg145Cys and alphaArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants betaArg263Leu, betaArg263Asn and betaArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that betaArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/K(m)) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the alphaArg145 mutants exhibited a 3-6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis-Menten complex accounts for the improved activity with beta-lactam substrates. Analysis of pH-activity profiles of wild-type enzyme and the betaArg263Lys mutant showed that betaArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which alphaArg145 is involved in binding of beta-lactam substrates and betaArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

Full Text

The Full Text of this article is available as a PDF (161.0 KB).

Selected References

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

  1. Alkema W. B., Floris R., Janssen D. B. The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases. Anal Biochem. 1999 Nov 1;275(1):47–53. doi: 10.1006/abio.1999.4300. [DOI] [PubMed] [Google Scholar]
  2. Alkema W. B., Hensgens C. M., Kroezinga E. H., de Vries E., Floris R., van der Laan J. M., Dijkstra B. W., Janssen D. B. Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies. Protein Eng. 2000 Dec;13(12):857–863. doi: 10.1093/protein/13.12.857. [DOI] [PubMed] [Google Scholar]
  3. Barbero J. L., Buesa J. M., González de Buitrago G., Méndez E., Péz-Aranda A., García J. L. Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila. Gene. 1986;49(1):69–80. doi: 10.1016/0378-1119(86)90386-0. [DOI] [PubMed] [Google Scholar]
  4. Brannigan J. A., Dodson G., Duggleby H. J., Moody P. C., Smith J. L., Tomchick D. R., Murzin A. G. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature. 1995 Nov 23;378(6555):416–419. doi: 10.1038/378416a0. [DOI] [PubMed] [Google Scholar]
  5. Done S. H., Brannigan J. A., Moody P. C., Hubbard R. E. Ligand-induced conformational change in penicillin acylase. J Mol Biol. 1998 Nov 27;284(2):463–475. doi: 10.1006/jmbi.1998.2180. [DOI] [PubMed] [Google Scholar]
  6. Duggleby H. J., Tolley S. P., Hill C. P., Dodson E. J., Dodson G., Moody P. C. Penicillin acylase has a single-amino-acid catalytic centre. Nature. 1995 Jan 19;373(6511):264–268. doi: 10.1038/373264a0. [DOI] [PubMed] [Google Scholar]
  7. Gololobov M. Y., Borisov I. L., Svedas V. K. Acyl group transfer by proteases forming an acylenzyme intermediate: kinetic model analysis (including hydrolysis of acylenzyme- nucleophile complex). J Theor Biol. 1989 Sep 22;140(2):193–204. doi: 10.1016/s0022-5193(89)80128-6. [DOI] [PubMed] [Google Scholar]
  8. Hewitt L., Kasche V., Lummer K., Lewis R. J., Murshudov G. N., Verma C. S., Dodson G. G., Wilson K. S. Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft. J Mol Biol. 2000 Sep 29;302(4):887–898. doi: 10.1006/jmbi.2000.4105. [DOI] [PubMed] [Google Scholar]
  9. Jackson S. E., Fersht A. R. Contribution of long-range electrostatic interactions to the stabilization of the catalytic transition state of the serine protease subtilisin BPN'. Biochemistry. 1993 Dec 21;32(50):13909–13916. doi: 10.1021/bi00213a021. [DOI] [PubMed] [Google Scholar]
  10. Kasche V., Lummer K., Nurk A., Piotraschke E., Rieks A., Stoeva S., Voelter W. Intramolecular autoproteolysis initiates the maturation of penicillin amidase from Escherichia coli. Biochim Biophys Acta. 1999 Aug 17;1433(1-2):76–86. doi: 10.1016/s0167-4838(99)00155-7. [DOI] [PubMed] [Google Scholar]
  11. Ljubijankić G., Konstantinović M., Glisin V. The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases. DNA Seq. 1992;3(3):195–200. doi: 10.3109/10425179209034017. [DOI] [PubMed] [Google Scholar]
  12. Margolin A. L., Svedas V. K., Berezin I. V. Substrate specificity of penicillin amidase from E. coli. Biochim Biophys Acta. 1980 Dec 4;616(2):283–289. doi: 10.1016/0005-2744(80)90145-x. [DOI] [PubMed] [Google Scholar]
  13. Martín L., Prieto M. A., Cortés E., García J. L. Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):287–292. doi: 10.1016/0378-1097(94)00510-x. [DOI] [PubMed] [Google Scholar]
  14. Morillas M., Goble M. L., Virden R. The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre. Biochem J. 1999 Feb 15;338(Pt 1):235–239. [PMC free article] [PubMed] [Google Scholar]
  15. Ménard R., Plouffe C., Laflamme P., Vernet T., Tessier D. C., Thomas D. Y., Storer A. C. Modification of the electrostatic environment is tolerated in the oxyanion hole of the cysteine protease papain. Biochemistry. 1995 Jan 17;34(2):464–471. doi: 10.1021/bi00002a010. [DOI] [PubMed] [Google Scholar]
  16. Ohashi H., Katsuta Y., Hashizume T., Abe S. N., Kajiura H., Hattori H., Kamei T., Yano M. Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus. Appl Environ Microbiol. 1988 Nov;54(11):2603–2607. doi: 10.1128/aem.54.11.2603-2607.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prabhune A. A., Sivaraman H. Evidence for involvement of arginyl residue at the catalytic site of penicillin acylase from Escherichia coli. Biochem Biophys Res Commun. 1990 Nov 30;173(1):317–322. doi: 10.1016/s0006-291x(05)81059-9. [DOI] [PubMed] [Google Scholar]
  18. Schumacher G., Sizmann D., Haug H., Buckel P., Böck A. Penicillin acylase from E. coli: unique gene-protein relation. Nucleic Acids Res. 1986 Jul 25;14(14):5713–5727. doi: 10.1093/nar/14.14.5713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sizmann D., Keilmann C., Böck A. Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105. Eur J Biochem. 1990 Aug 28;192(1):143–151. doi: 10.1111/j.1432-1033.1990.tb19207.x. [DOI] [PubMed] [Google Scholar]
  20. Verhaert R. M., Riemens A. M., van der Laan J. M., van Duin J., Quax W. J. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis. Appl Environ Microbiol. 1997 Sep;63(9):3412–3418. doi: 10.1128/aem.63.9.3412-3418.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Warshel A. Electrostatic origin of the catalytic power of enzymes and the role of preorganized active sites. J Biol Chem. 1998 Oct 16;273(42):27035–27038. doi: 10.1074/jbc.273.42.27035. [DOI] [PubMed] [Google Scholar]
  22. Zafaralla G., Manavathu E. K., Lerner S. A., Mobashery S. Elucidation of the role of arginine-244 in the turnover processes of class A beta-lactamases. Biochemistry. 1992 Apr 21;31(15):3847–3852. doi: 10.1021/bi00130a016. [DOI] [PubMed] [Google Scholar]

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

RESOURCES