Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus

  • Sanghamitra Mitra
  • George Sheppard
  • Jieyi Wang
  • Brian Bennett
  • Richard C. Holz
Original Paper


Methionine aminopeptidases (MetAPs) represent a unique class of protease that is capable of the hydrolytic removal of an N-terminal methionine residue from nascent polypeptide chains. MetAPs are physiologically important enzymes; hence, there is considerable interest in developing inhibitors that can be used as antiangiogenic and antimicrobial agents. A detailed kinetic and spectroscopic study has been performed to probe the binding of a triazole-based inhibitor and a bestatin-based inhibitor to both Mn(II)- and Co(II)-loaded type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs. Both inhibitors were found to be moderate competitive inhibitors. The triazole-type inhibitor was found to interact with both active-site metal ions, while the bestatin-type inhibitor was capable of switching its mode of binding depending on the metal in the active site and the type of MetAP enzyme.


Hydrolysis Manganese Electron paramagnetic resonance Antibiotics Electronic absorption 



This work was supported by the National Science Foundation (CHE-0652981, R.C.H.) and the National Institutes of Health (AI056231, B.B.). The Bruker Elexsys spectrometer was purchased by the Medical College of Wisconsin and is supported with funds from the National Institutes of Health (EB001980, B.B.).


  1. 1.
    Lowther TW, Zhang Y, Sampson PB, Honek JF, Matthews BW (1999) Insights into the mechanism of E. coli methionine aminopeptidase from the structural analysis of reaction products and phosphorous-based transition state analogs. Biochemistry 38:14810–14819PubMedCrossRefGoogle Scholar
  2. 2.
    Lowther WT, Matthews BW (2002) Metalloaminopeptidases: common functional themes in disparate structural surroundings. Chem Rev 102:4581–4607PubMedCrossRefGoogle Scholar
  3. 3.
    Griffith EC, Su Z, Turk BE, Chen S, Chang Y-H, Wu Z, Biemann K, Liu JO (1997) Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin. Chem Biol 4:461–471PubMedCrossRefGoogle Scholar
  4. 4.
    Selvakumar P, Lakshmikuttyamma A, Kanthan R, Kanthan SC, Dimmock JR, Sharma RK (2004) High expression of methionine aminopeptidase 2 in human colorectal adenocarcinomas. Clin Cancer Res 10:2771–2775PubMedCrossRefGoogle Scholar
  5. 5.
    Selvakumar P, Lakshmikuttyamma A, Lawman Z, Bonham K, Dimmock JR, Sharma RK (2004) Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29. Biochem Biophys Res Commun 322:1012–1017PubMedCrossRefGoogle Scholar
  6. 6.
    Bradshaw RA (1989) Protein translocation and turnover in eukaryotic cells. Trends Biochem Sci 14:276–279PubMedCrossRefGoogle Scholar
  7. 7.
    Meinnel T, Mechulam Y, Blanquet S (1993) Methionine as translation start signal—a review of the enzymes of the pathway in Escherichia coli. Biochimie 75:1061–1075PubMedCrossRefGoogle Scholar
  8. 8.
    Bradshaw RA, Brickey WW, Walker KW (1998) N-terminal processing: The methionine aminopeptidase and N α-acetyl transferase families. Trends Biochem Sci 23:263–267PubMedCrossRefGoogle Scholar
  9. 9.
    Arfin SM, Bradshaw RA (1988) Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry 27(21):7979–7984PubMedCrossRefGoogle Scholar
  10. 10.
    Taunton J (1997) How to starve a tumor. Chem Biol 4:493–496PubMedCrossRefGoogle Scholar
  11. 11.
    Wang J, Tucker LA, Stavropoulos J, Zhang Q, Wang Y-C, Bukofzer G, Niquette A, Meulbroek JA, Barnes DM, Shen J, Bouska J, Donawho C, Sheppard GS, Bell RL (2008) Correlation of tumor growth suppression and methionine aminopetidase-2 activity blockade using an orally active inhibitor. Proc Natl Acad Sci USA 105:1838–1843PubMedCrossRefGoogle Scholar
  12. 12.
    Tucker LA, Zhang Q, Sheppard GS, Lou P, Jiang F, McKeegan E, Lesniewski R, Davidsen SK, Bell RL, Wang J (2008) Ectopic expression of methionine aminopeptidase-2 causes cell transformation and stimulates proliferation. Oncogene 27:3967–3976PubMedCrossRefGoogle Scholar
  13. 13.
    Benny O, Fainaru O, Adini A, Cassiola F, Bazinet L, Adini I, Pravda E, Nahmias Y, Koirala S, Corfas G, D’Amato RJ, Folkman J (2008) An orally delivered small-molecule formulation with antiangiogenic and anticancer activity. Nat Biotechnol 26:799–807PubMedCrossRefGoogle Scholar
  14. 14.
    Satchi-Fainaro R, Mamluk R, Wang L, Short SM, Nagy JA, Feng D, Dvorak AM, Dvorak HF, Puder M, Mukhopadhyay D, Folkman J (2005) Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin. Cancer Cell 7:251–261PubMedCrossRefGoogle Scholar
  15. 15.
    Zhang P, Nicholson DE, Bujnicki JM, Su X, Brendle JJ, Ferdig M, Kyle DE, Milhous WK, Chiang PK (2002) Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for malaria and leishmaniasis. J Biomed Sci 9:34–40PubMedCrossRefGoogle Scholar
  16. 16.
    Douangamath A, Dale GE, D’Arcy A, Almstetter M, Eckl R, Frutos-Hoener A, Henkel B, Illgen K, Nerdinger S, Schulz H, MacSweeney A, Thormann M, Treml A, Pierau S, Wadman S, Oefner C (2004) Crystal structures of Staphylococcus aureus methionine aminopeptidase complexed with keto heterocycle and aminoketone inhibitors reveal the formation of a tetrahedral intermediate. J Med Chem 47:1325–1328PubMedCrossRefGoogle Scholar
  17. 17.
    Tahirov TH, Oki H, Tsukihara T, Ogasahara K, Yutani K, Ogata K, Izu Y, Tsunasawa S, Kato I (1998) Crystal structure of the methionine aminopeptidase from the hyperthermophile, Pyrococcus furiosus. J Mol Biol 284:101–124PubMedCrossRefGoogle Scholar
  18. 18.
    Lowther WT, Orville AM, Madden DT, Lim S, Rich DH, Matthews BW (1999) Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant and inhibited enzymes for the mechanism of catalysis. Biochemistry 38:7678–7688PubMedCrossRefGoogle Scholar
  19. 19.
    Roderick LS, Matthews BW (1993) Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme. Biochemistry 32:3907–3912PubMedCrossRefGoogle Scholar
  20. 20.
    Liu S, Widom J, Kemp CW, Crews CM, Clardy J (1998) Structure of the human methionine aminopeptidase-2 complexed with fumagillin. Science 282:1324–1327PubMedCrossRefGoogle Scholar
  21. 21.
    Spraggon G, Schwarzenbacher R, Kreusch A, McMullan D, Brinen LS, Canaves JM, Dai X, Deacon AM, Elsliger MA, Eshagi S, Floyd R, Godzik A, Grittini C, Grzechnik SK, Jaroszewski L, Karlak C, Klock HE, Koesema E, Kovarik JS, Kuhn P, McPhillips TM, Miller MD, Morse A, Moy K, Ouyang J, Page R, Quijano K, Rezezadeh F, Robb A, Sims E, Stevens RC, van den Bedem H, Velasquez J, Vincent J, von Delft F, Wang X, West B, Wolf G, Xu Q, Hodgson KO, Wooley J, Lesley SA, Wilson IA (2004) Crystal structure of a methionine aminopeptidase (TM1478) from Thermotoga maritima at 1.9 Å resolution. Proteins 56:396–400PubMedCrossRefGoogle Scholar
  22. 22.
    Ye QZ, Xie SX, Ma ZQ, Huang M, Hanzlik RP (2006) Structural basis of catalysis by monometalated methionine aminopeptidase. Proc Natl Acad Sci USA 103:9470–9475PubMedCrossRefGoogle Scholar
  23. 23.
    D’souza VM, Bennett B, Copik AJ, Holz RC (2000) Characterization of the divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli. Biochemistry 39:3817–3826PubMedCrossRefGoogle Scholar
  24. 24.
    Cosper NJ, D’souza V, Scott R, Holz RC (2001) Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease. Biochemistry 40:13302–13309PubMedCrossRefGoogle Scholar
  25. 25.
    Wang J, Sheppard GS, Lou P, Kawai M, Park C, Egan DA, Schneider A, Bouska J, Lesniewski R, Henkin J (2003) Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese. Biochemistry 42:5035–5042PubMedCrossRefGoogle Scholar
  26. 26.
    Chai SC, Wang W-L, Ye Q-Z (2008) Fe(II) is the native cofactor for Escherichia coli methionine aminopeptidase J Biol Chem 283 (in press)Google Scholar
  27. 27.
    D’souza VM, Holz RC (1999) The methionyl aminopeptidase from Escherichia coli is an iron(II) containing enzyme. Biochemistry 38:11079–11085PubMedCrossRefGoogle Scholar
  28. 28.
    Meng L, Ruebush S, D’souza VM, Copik AJ, Tsunasawa S, Holz RC (2002) Overexpression and divalent metal binding studies for the methionyl aminopeptidase from Pyrococcus furiosus. Biochemistry 41:7199–7208PubMedCrossRefGoogle Scholar
  29. 29.
    Walker KW, Bradshaw RA (1998) Yeast methionine aminopeptidase I can utilize either Zn(II) or Co(II) as a cofactor: A case of mistaken identity. Protein Sci 7:2684–2687PubMedCrossRefGoogle Scholar
  30. 30.
    Copik AJ, Swierczek SI, Lowther WT, D’souza V, Matthews BW, Holz RC (2003) Kinetic and spectroscopic characterization of the H178A mutant of the methionyl aminopeptidase from Escherichia coli. Biochemistry 42:6283–6292PubMedCrossRefGoogle Scholar
  31. 31.
    Stamper C, Bennett B, Edwards T, Holz RC, Ringe D, Petsko G (2001) Inhibition of the aminopeptidase from Aeromonas proteolytica by l-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis. Biochemistry 40:7034–7046CrossRefGoogle Scholar
  32. 32.
    Stamper C, Bienvenue D, Moulin A, Bennett B, Ringe D, Petsko G, Holz RC (2004) Spectroscopic and X-ray crystallographic characterization of the bestatin bound form of the aminopeptidase from Aeromonas proteolytica. Biochemistry 43:9620–9628PubMedCrossRefGoogle Scholar
  33. 33.
    Kumar A, Periyannan GR, Narayanan B, Kittell AW, Kim J-J, Bennett B (2007) Experimental evidence for a metallohydrolase mechanism in which the neucleophile is not delivered by a metal ion: EPR spectrokinetic and structural studies of aminopeptidase from Vibrio proteolyticus. Biochem J 403:527–536PubMedCrossRefGoogle Scholar
  34. 34.
    Sin N, Meng L, Wang MQW, Wen JJ, Bornmann WG, Crews CM (1997) The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. Proc Natl Acad Sci USA 94:6099–6103PubMedCrossRefGoogle Scholar
  35. 35.
    Swierczek K, Copik AJ, Swierczek SI, Holz RC (2005) Molecular discrimination of type-I over type-II methionyl aminopeptidases. Biochemistry 44:12049–12056PubMedCrossRefGoogle Scholar
  36. 36.
    Luo Q-L, Li J-Y, Liu Z-Y, Chen L-L, Li J, Qian Z, Shen Q, Li Y, Lushington GH, Ye Q-Z, Nan F-J (2003) J Med Chem 46:2631–2640PubMedCrossRefGoogle Scholar
  37. 37.
    Bradshaw R, Yi E (2002) Methionine aminopeptidases and angiogenesis. Essays Biol Med 38:65–78Google Scholar
  38. 38.
    Lowther WT, Matthews BW (2000) Structure and function of the methionine aminopeptidases. Biochim Biophys Acta 1477:157–167PubMedGoogle Scholar
  39. 39.
    Addlagatta A, Hu X, Liu JO, Matthews BW (2005) Structural basis for the functional differences between type I and type II human methionine aminopeptidases. Biochemistry 44:14741–14749PubMedCrossRefGoogle Scholar
  40. 40.
    Bertini I, Luchinat C (1984) High-spin cobalt(II) as a probe for the investigation of metalloproteins. Adv Inorg Biochem 6:71–111PubMedGoogle Scholar
  41. 41.
    Bennett B, Holz RC (1997) EPR studies on the mono- and dicobalt(II)-substituted forms of the aminopeptidase from Aeromonas proteolytica. Insight into the catalytic mechanism of dinuclear hydrolases. J Am Chem Soc 119:1923–1933CrossRefGoogle Scholar
  42. 42.
    Bennett B, Holz RC (1997) Spectroscopically distinct cobalt(II) sites in heterodimetallic forms of the aminopeptidase from Aeromonas proteolytica: characterization of substrate binding. Biochemistry 36:9837–9846PubMedCrossRefGoogle Scholar
  43. 43.
    Gavrilova AL, Bosnich B (2004) Principles of mononucleating and bionucleating ligand design. Chem Rev 104:349–383PubMedCrossRefGoogle Scholar
  44. 44.
    Escuer A, Vicente R, Mernari B, El Gueddi A, Pierrot M (1997) Syntheses, structure, and magnetic behavior of two new nickel(II) and cobalt(II) dinuclear complexes with 1, 4-dicarboxylatopyridazine. MO calculations of the superexchange pathway through the pyridazine bridge. Inorg Chem 36:2511–2516CrossRefGoogle Scholar
  45. 45.
    Yoo HS, Lim JH, Kang JS, Koh EK, Hong CS (2007) Triazole-bridged magnetic M(II) assemblies (M = Co, Ni) capped with the end-on terephthalate dianion involving multi-intermolecular contacts. Polyhedron 26:4383–4388CrossRefGoogle Scholar
  46. 46.
    Reed GH, Markham GD (1984) Biol Magn Reson 6:73–142Google Scholar
  47. 47.
    McGregor WC, Swierczek SI, Bennett B, Holz RC (2007) Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli. J Biol Inorg Chem 12:603–613PubMedCrossRefGoogle Scholar
  48. 48.
    Sheppard GS, Wang J, Kawai M, BaMaung NY, Craig RA, Erickson SA, Lynch L, Patel J, Yang F, Searle XB, Lou P, Park C, Kim KH, Henkin J, Lesniewski R (2004) Bioorg Med Chem Lett 14:865–868PubMedCrossRefGoogle Scholar
  49. 49.
    Kallander LS, Lu Q, Chen W, Tomaszek T, Yang G, Tew D, Meek TD, Hofmann GA, Schulz-Pritchard CK, Smith WW, Janson CA, Ryan MD, Zhang G-F, Johanson KO, Kirkpatrick RB, Ho TF, Fisher PW, Mattern MR, Johnson RK, Hansbury MJ, Winkler JD, Ward KW, Veber DF, Thompson SK (2005) 4-Aryl-1, 2, 3-triazole: a novel template for a reversible methionine aminopeptidase 2 inhibitor, optimized to inhibit angiogenesis in vivo. J Med Chem 48:5644–5647PubMedCrossRefGoogle Scholar
  50. 50.
    Oefner C, Douangamath A, D’Arcy AHS, Mareque D, Mac Sweeney A, Padilla J, Pierau S, Schulz H, Thormann M, Wadman S, Dale GE (2003) J Mol Biol 332:13–21PubMedCrossRefGoogle Scholar
  51. 51.
    Marino JP, Fisher PW, Hofmann GA, Kirkpatrick RB, Janson CA, Johnson RK, Ma C, Mattern M, Meek TD, Ryan MD, Schulz C, Smith WW, Tew DG, Tomazek TA, Veber DF, Xiong WC, Yamamoto Y, Yamashita K, Yang G, Thompson SK (2007) Highly potent inhibitors of methionine aminopeptidase-2 based on a 1, 2, 4-triazole pharmacophore. J Med Chem 50:3777–3785PubMedCrossRefGoogle Scholar
  52. 52.
    Xie S-X, Huang W-J, Ma Z-Q, Huang M, Hanzlik RP, Ye Q-Z (2006) Acta Crystallogr D 62:425–432PubMedCrossRefGoogle Scholar
  53. 53.
    Sigel H, McCormick DB (1970) Acc Chem Res 3:201–208CrossRefGoogle Scholar
  54. 54.
    Larrabee JA, Leung CH, Moore R, Thamrong-nawasawat T, Wessler BH (2004) Magnetic circular dichroism and cobalt(II) binding equilibrium studies of Escherichia coli methionyl aminopeptidase. J Am Chem Soc 126:12316–12324PubMedCrossRefGoogle Scholar
  55. 55.
    Hu XV, Chen X, Han KC, Mildvan AS, Liu JO (2007) Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases. Biochemistry 46:12833–12843PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2009

Authors and Affiliations

  • Sanghamitra Mitra
    • 1
    • 4
  • George Sheppard
    • 3
  • Jieyi Wang
    • 3
  • Brian Bennett
    • 2
  • Richard C. Holz
    • 1
  1. 1.Department of ChemistryLoyola University-ChicagoChicagoUSA
  2. 2.Department of Biophysics, The National Biomedical EPR CenterMedical College of WisconsinMilwaukeeUSA
  3. 3.Cancer Research, Global Pharmaceutical R&DAbbott LaboratoriesAbbott ParkUSA
  4. 4.Department of ChemistryBoston UniversityBostonUSA

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