Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Aug 1;349(Pt 3):813–819. doi: 10.1042/bj3490813

Role of arg-410 and tyr-411 in human serum albumin for ligand binding and esterase-like activity.

H Watanabe 1, S Tanase 1, K Nakajou 1, T Maruyama 1, U Kragh-Hansen 1, M Otagiri 1
PMCID: PMC1221209  PMID: 10903143

Abstract

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411.

Full Text

The Full Text of this article is available as a PDF (148.4 KB).

Selected References

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

  1. Awad-Elkarim A., Means G. E. The reactivity of p-nitrophenyl acetate with serum albumins. Comp Biochem Physiol B. 1988;91(2):267–272. doi: 10.1016/0305-0491(88)90141-1. [DOI] [PubMed] [Google Scholar]
  2. Chen R. F. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967 Jan 25;242(2):173–181. [PubMed] [Google Scholar]
  3. Chuang V. T., Kuniyasu A., Nakayama H., Matsushita Y., Hirono S., Otagiri M. Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety. Biochim Biophys Acta. 1999 Sep 14;1434(1):18–30. doi: 10.1016/s0167-4838(99)00174-0. [DOI] [PubMed] [Google Scholar]
  4. Curry S., Mandelkow H., Brick P., Franks N. Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nat Struct Biol. 1998 Sep;5(9):827–835. doi: 10.1038/1869. [DOI] [PubMed] [Google Scholar]
  5. Dubois N., Lapicque F., Magdalou J., Abiteboul M., Netter P. Stereoselective binding of the glucuronide of ketoprofen enantiomers to human serum albumin. Biochem Pharmacol. 1994 Nov 1;48(9):1693–1699. doi: 10.1016/0006-2952(94)90453-7. [DOI] [PubMed] [Google Scholar]
  6. Fehske K. J., Müller W. E., Wollert U. A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin. Biochim Biophys Acta. 1979 Apr 25;577(2):346–359. doi: 10.1016/0005-2795(79)90038-2. [DOI] [PubMed] [Google Scholar]
  7. Fehske K. J., Schläfer U., Wollert U., Müller W. E. Characterization of an important drug binding area on human serum albumin including the high-affinity binding sites of warfarin and azapropazone. Mol Pharmacol. 1982 Mar;21(2):387–393. [PubMed] [Google Scholar]
  8. Galliano M., Watkins S., Madison J., Putnam F. W., Kragh-Hansen U., Cesati R., Minchiotti L. Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA. Eur J Biochem. 1998 Jan 15;251(1-2):329–334. doi: 10.1046/j.1432-1327.1998.2510329.x. [DOI] [PubMed] [Google Scholar]
  9. Gambhir K. K., McMenamy R. H. Location of the indole binding site in human serum albumin. Characterization of major cyanogen bromide fragments with respect to affinity labeling positions. J Biol Chem. 1973 Mar 25;248(6):1956–1960. [PubMed] [Google Scholar]
  10. He X. M., Carter D. C. Atomic structure and chemistry of human serum albumin. Nature. 1992 Jul 16;358(6383):209–215. doi: 10.1038/358209a0. [DOI] [PubMed] [Google Scholar]
  11. Ho J. X., Holowachuk E. W., Norton E. J., Twigg P. D., Carter D. C. X-ray and primary structure of horse serum albumin (Equus caballus) at 0.27-nm resolution. Eur J Biochem. 1993 Jul 1;215(1):205–212. doi: 10.1111/j.1432-1033.1993.tb18024.x. [DOI] [PubMed] [Google Scholar]
  12. Kosa T., Maruyama T., Otagiri M. Species differences of serum albumins: I. Drug binding sites. Pharm Res. 1997 Nov;14(11):1607–1612. doi: 10.1023/a:1012138604016. [DOI] [PubMed] [Google Scholar]
  13. Kragh-Hansen U., Brennan S. O., Galliano M., Sugita O. Binding of warfarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations. Mol Pharmacol. 1990 Feb;37(2):238–242. [PubMed] [Google Scholar]
  14. Kragh-Hansen U. Molecular aspects of ligand binding to serum albumin. Pharmacol Rev. 1981 Mar;33(1):17–53. [PubMed] [Google Scholar]
  15. Kurono Y., Kushida I., Tanaka H., Ikeda K. Esterase-like activity of human serum albumin. VIII. Reaction with amino acid p-nitrophenyl esters. Chem Pharm Bull (Tokyo) 1992 Aug;40(8):2169–2172. doi: 10.1248/cpb.40.2169. [DOI] [PubMed] [Google Scholar]
  16. MURACHI T. A general reaction of diisopropylphosphorofluoridate with proteins without direct effect on enzymic activities. Biochim Biophys Acta. 1963 Apr 2;71:239–241. doi: 10.1016/0006-3002(63)91021-7. [DOI] [PubMed] [Google Scholar]
  17. Maruyama K., Nishigori H., Iwatsuru M. Characterization of the benzodiazepine binding site (diazepam site) on human serum albumin. Chem Pharm Bull (Tokyo) 1985 Nov;33(11):5002–5012. doi: 10.1248/cpb.33.5002. [DOI] [PubMed] [Google Scholar]
  18. Maruyama T., Lin C. C., Yamasaki K., Miyoshi T., Imai T., Yamasaki M., Otagiri M. Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group. Biochem Pharmacol. 1993 Mar 9;45(5):1017–1026. doi: 10.1016/0006-2952(93)90245-r. [DOI] [PubMed] [Google Scholar]
  19. Means G. E., Bender M. L. Acetylation of human serum albumin by p-nitrophenyl acetate. Biochemistry. 1975 Nov 4;14(22):4989–4994. doi: 10.1021/bi00693a031. [DOI] [PubMed] [Google Scholar]
  20. Means G. E., Wu H. L. The reactive tyrosine residue of human serum albumin: characterization of its reaction with diisopropylfluorophosphate. Arch Biochem Biophys. 1979 May;194(2):526–530. doi: 10.1016/0003-9861(79)90647-7. [DOI] [PubMed] [Google Scholar]
  21. Minghetti P. P., Ruffner D. E., Kuang W. J., Dennison O. E., Hawkins J. W., Beattie W. G., Dugaiczyk A. Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4. J Biol Chem. 1986 May 25;261(15):6747–6757. [PubMed] [Google Scholar]
  22. Müller W., Wollert U. Influence of pH on the benzodiazepine-human serum albumin complex. Circular dichroism studies. Naunyn Schmiedebergs Arch Pharmacol. 1974;283(1):67–82. doi: 10.1007/BF00500146. [DOI] [PubMed] [Google Scholar]
  23. Müller W., Wollert U. Interactions of benzodiazepines with human serum albumin. Circular dichroism studies. Naunyn Schmiedebergs Arch Pharmacol. 1973;278(3):301–312. doi: 10.1007/BF00500291. [DOI] [PubMed] [Google Scholar]
  24. Narazaki R., Watanabe H., Maruyama T., Suenaga A., Otagiri M. An immunological method for the detection of captopril-protein conjugate. Arch Toxicol. 1998 Mar;72(4):203–206. doi: 10.1007/s002040050489. [DOI] [PubMed] [Google Scholar]
  25. Ozeki Y., Kurono Y., Yotsuyanagi T., Ikeda K. Effects of drug binding on the esterase activity of human serum albumin: inhibition modes and binding sites of anionic drugs. Chem Pharm Bull (Tokyo) 1980 Feb;28(2):535–540. doi: 10.1248/cpb.28.535. [DOI] [PubMed] [Google Scholar]
  26. Petersen C. E., Ha C. E., Harohalli K., Park D., Bhagavan N. V. Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A. Biochemistry. 1997 Jun 10;36(23):7012–7017. doi: 10.1021/bi970225v. [DOI] [PubMed] [Google Scholar]
  27. Rahman M. H., Maruyama T., Okada T., Imai T., Otagiri M. Study of interaction of carprofen and its enantiomers with human serum albumin--II. Stereoselective site-to-site displacement of carprofen by ibuprofen. Biochem Pharmacol. 1993 Nov 17;46(10):1733–1740. doi: 10.1016/0006-2952(93)90577-j. [DOI] [PubMed] [Google Scholar]
  28. Sjödin T., Hansson R., Sjöholm I. Isolation and identification of a trypsin-resistant fragment of human serum albumin with bilirubin- and drug-binding properties. Biochim Biophys Acta. 1977 Sep 27;494(1):61–75. doi: 10.1016/0005-2795(77)90135-0. [DOI] [PubMed] [Google Scholar]
  29. Sjödin T., Roosdorp N., Sjöholm I. Studies on the binding of benzodiazepines to human serum albumin by circular dichroism measurements. Biochem Pharmacol. 1976 Oct 1;25(19):2131–2140. doi: 10.1016/0006-2952(76)90124-6. [DOI] [PubMed] [Google Scholar]
  30. Sjöholm I., Ljungstedt I. Studies on the tryptophan and drug-binding properties of human serum albumin fragments by affinity chromatography and circular dichroism measurements. J Biol Chem. 1973 Dec 25;248(24):8434–8441. [PubMed] [Google Scholar]
  31. Sudlow G., Birkett D. J., Wade D. N. Further characterization of specific drug binding sites on human serum albumin. Mol Pharmacol. 1976 Nov;12(6):1052–1061. [PubMed] [Google Scholar]
  32. Sudlow G., Birkett D. J., Wade D. N. The characterization of two specific drug binding sites on human serum albumin. Mol Pharmacol. 1975 Nov;11(6):824–832. [PubMed] [Google Scholar]
  33. Yamamoto Y., Taniyama Y., Kikuchi M., Ikehara M. Engineering of the hydrophobic segment of the signal sequence for efficient secretion of human lysozyme by Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1987 Dec 16;149(2):431–436. doi: 10.1016/0006-291x(87)90385-8. [DOI] [PubMed] [Google Scholar]
  34. Zandomeneghi M. Circular dichroism of ketoprofen complexed to serum albumins: conformational selection by the protein: a novel optical purity determination technique. Chirality. 1995;7(6):446–451. doi: 10.1002/chir.530070610. [DOI] [PubMed] [Google Scholar]

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

RESOURCES