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. 1997 Nov 1;327(Pt 3):883–889. doi: 10.1042/bj3270883

A tyrosine residue essential for catalytic activity in aminopeptidase A.

G Vazeux 1, X Iturrioz 1, P Corvol 1, C Llorens-Cortès 1
PMCID: PMC1218871  PMID: 9581570

Abstract

Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.

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Selected References

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  1. Aoyagi T., Tobe H., Kojima F., Hamada M., Takeuchi T., Umezawa H. Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes. J Antibiot (Tokyo) 1978 Jun;31(6):636–638. doi: 10.7164/antibiotics.31.636. [DOI] [PubMed] [Google Scholar]
  2. Beaumont A., O'Donohue M. J., Paredes N., Rousselet N., Assicot M., Bohuon C., Fournié-Zaluski M. C., Roques B. P. The role of histidine 231 in thermolysin-like enzymes. A site-directed mutagenesis study. J Biol Chem. 1995 Jul 14;270(28):16803–16808. doi: 10.1074/jbc.270.28.16803. [DOI] [PubMed] [Google Scholar]
  3. Blomster M., Wetterholm A., Mueller M. J., Haeggström J. Z. Evidence for a catalytic role of tyrosine 383 in the peptidase reaction of leukotriene A4 hydrolase. Eur J Biochem. 1995 Aug 1;231(3):528–534. doi: 10.1111/j.1432-1033.1995.0528d.x. [DOI] [PubMed] [Google Scholar]
  4. Chauvel E. N., Coric P., Llorens-Cortès C., Wilk S., Roques B. P., Fournié-Zaluski M. C. Investigation of the active site of aminopeptidase A using a series of new thiol-containing inhibitors. J Med Chem. 1994 Apr 29;37(9):1339–1346. doi: 10.1021/jm00035a014. [DOI] [PubMed] [Google Scholar]
  5. Chauvel E. N., Llorens-Cortès C., Coric P., Wilk S., Roques B. P., Fournié-Zaluski M. C. Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols. J Med Chem. 1994 Sep 2;37(18):2950–2957. doi: 10.1021/jm00044a016. [DOI] [PubMed] [Google Scholar]
  6. Dion N., Le Moual H., Crine P., Boileau G. Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. FEBS Lett. 1993 Mar 8;318(3):301–304. doi: 10.1016/0014-5793(93)80533-z. [DOI] [PubMed] [Google Scholar]
  7. Fersht A. R., Shi J. P., Knill-Jones J., Lowe D. M., Wilkinson A. J., Blow D. M., Brick P., Carter P., Waye M. M., Winter G. Hydrogen bonding and biological specificity analysed by protein engineering. Nature. 1985 Mar 21;314(6008):235–238. doi: 10.1038/314235a0. [DOI] [PubMed] [Google Scholar]
  8. Gardell S. J., Craik C. S., Hilvert D., Urdea M. S., Rutter W. J. Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis. Nature. 1985 Oct 10;317(6037):551–555. doi: 10.1038/317551a0. [DOI] [PubMed] [Google Scholar]
  9. Grams F., Dive V., Yiotakis A., Yiallouros I., Vassiliou S., Zwilling R., Bode W., Stöcker W. Structure of astacin with a transition-state analogue inhibitor. Nat Struct Biol. 1996 Aug;3(8):671–675. doi: 10.1038/nsb0896-671. [DOI] [PubMed] [Google Scholar]
  10. Healy D. P., Wilk S. Localization of immunoreactive glutamyl aminopeptidase in rat brain. II. Distribution and correlation with angiotensin II. Brain Res. 1993 Mar 26;606(2):295–303. doi: 10.1016/0006-8993(93)90997-2. [DOI] [PubMed] [Google Scholar]
  11. Helene A., Beaumont A., Roques B. P. Functional residues at the active site of aminopeptidase N. Eur J Biochem. 1991 Mar 14;196(2):385–393. doi: 10.1111/j.1432-1033.1991.tb15828.x. [DOI] [PubMed] [Google Scholar]
  12. Herlitze S., Koenen M. A general and rapid mutagenesis method using polymerase chain reaction. Gene. 1990 Jul 2;91(1):143–147. doi: 10.1016/0378-1119(90)90177-s. [DOI] [PubMed] [Google Scholar]
  13. Hooper N. M. Families of zinc metalloproteases. FEBS Lett. 1994 Oct 31;354(1):1–6. doi: 10.1016/0014-5793(94)01079-x. [DOI] [PubMed] [Google Scholar]
  14. Jongeneel C. V., Bouvier J., Bairoch A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 1989 Jan 2;242(2):211–214. doi: 10.1016/0014-5793(89)80471-5. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lejczak B., De Choszczak M. P., Kafarski P. Inhibition of aminopeptidases by phosphonic acid and phosphinic acid analogues of aspartic and glutamic acids. J Enzyme Inhib. 1993;7(2):97–103. doi: 10.3109/14756369309040752. [DOI] [PubMed] [Google Scholar]
  18. Li L., Wang J., Cooper M. D. cDNA cloning and expression of human glutamyl aminopeptidase (aminopeptidase A). Genomics. 1993 Sep;17(3):657–664. doi: 10.1006/geno.1993.1386. [DOI] [PubMed] [Google Scholar]
  19. Lojda Z., Gossrau R. Study on aminopeptidase A. Histochemistry. 1980;67(3):267–290. doi: 10.1007/BF00692761. [DOI] [PubMed] [Google Scholar]
  20. Medina J. F., Wetterholm A., Rådmark O., Shapiro R., Haeggström J. Z., Vallee B. L., Samuelsson B. Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7620–7624. doi: 10.1073/pnas.88.17.7620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Migaud M., Durieux C., Viereck J., Soroca-Lucas E., Fournié-Zaluski M. C., Roques B. P. The in vivo metabolism of cholecystokinin (CCK-8) is essentially ensured by aminopeptidase A. Peptides. 1996;17(4):601–607. doi: 10.1016/0196-9781(96)00036-8. [DOI] [PubMed] [Google Scholar]
  22. Mueller M. J., Samuelsson B., Haeggström J. Z. Chemical modification of leukotriene A4 hydrolase. Indications for essential tyrosyl and arginyl residues at the active site. Biochemistry. 1995 Mar 21;34(11):3536–3543. doi: 10.1021/bi00011a007. [DOI] [PubMed] [Google Scholar]
  23. Nagatsu I., Nagatsu T., Yamamoto T., Glenner G. G., Mehl J. W. Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme. Biochim Biophys Acta. 1970 Feb 11;198(2):255–270. doi: 10.1016/0005-2744(70)90058-6. [DOI] [PubMed] [Google Scholar]
  24. Nanus D. M., Engelstein D., Gastl G. A., Gluck L., Vidal M. J., Morrison M., Finstad C. L., Bander N. H., Albino A. P. Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7069–7073. doi: 10.1073/pnas.90.15.7069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ondetti M. A., Rubin B., Cushman D. W. Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. Science. 1977 Apr 22;196(4288):441–444. doi: 10.1126/science.191908. [DOI] [PubMed] [Google Scholar]
  26. Orning L., Gierse J. K., Fitzpatrick F. A. The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity. J Biol Chem. 1994 Apr 15;269(15):11269–11273. [PubMed] [Google Scholar]
  27. Orning L., Krivi G., Fitzpatrick F. A. Leukotriene A4 hydrolase. Inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes. J Biol Chem. 1991 Jan 25;266(3):1375–1378. [PubMed] [Google Scholar]
  28. Phillips M. A., Fletterick R., Rutter W. J. Arginine 127 stabilizes the transition state in carboxypeptidase. J Biol Chem. 1990 Nov 25;265(33):20692–20698. [PubMed] [Google Scholar]
  29. Roderick S. L., Fournie-Zaluski M. C., Roques B. P., Matthews B. W. Thiorphan and retro-thiorphan display equivalent interactions when bound to crystalline thermolysin. Biochemistry. 1989 Feb 21;28(4):1493–1497. doi: 10.1021/bi00430a011. [DOI] [PubMed] [Google Scholar]
  30. Schalk C., d'Orchymont H., Jauch M. F., Tarnus C. 3-Amino-2-tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-M (EC 3.4.11.2). Arch Biochem Biophys. 1994 May 15;311(1):42–46. doi: 10.1006/abbi.1994.1206. [DOI] [PubMed] [Google Scholar]
  31. Song L., Ye M., Troyanovskaya M., Wilk E., Wilk S., Healy D. P. Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization. Am J Physiol. 1994 Oct;267(4 Pt 2):F546–F557. doi: 10.1152/ajprenal.1994.267.4.F546. [DOI] [PubMed] [Google Scholar]
  32. Tieku S., Hooper N. M. Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme. Biochem Pharmacol. 1992 Nov 3;44(9):1725–1730. doi: 10.1016/0006-2952(92)90065-Q. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vazeux G., Wang J., Corvol P., Llorens-Cortès C. Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A. J Biol Chem. 1996 Apr 12;271(15):9069–9074. doi: 10.1074/jbc.271.15.9069. [DOI] [PubMed] [Google Scholar]
  34. Wang J., Cooper M. D. Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1222–1226. doi: 10.1073/pnas.90.4.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilk S., Thurston L. S. Inhibition of angiotensin III formation by thiol derivatives of acidic amino acids. Neuropeptides. 1990 Jul;16(3):163–168. doi: 10.1016/0143-4179(90)90129-m. [DOI] [PubMed] [Google Scholar]
  36. Wu Q., Lahti J. M., Air G. M., Burrows P. D., Cooper M. D. Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc Natl Acad Sci U S A. 1990 Feb;87(3):993–997. doi: 10.1073/pnas.87.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu Q., Li L., Cooper M. D., Pierres M., Gorvel J. P. Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):676–680. doi: 10.1073/pnas.88.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zini S., Fournie-Zaluski M. C., Chauvel E., Roques B. P., Corvol P., Llorens-Cortes C. Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11968–11973. doi: 10.1073/pnas.93.21.11968. [DOI] [PMC free article] [PubMed] [Google Scholar]

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