JBIC Journal of Biological Inorganic Chemistry

, Volume 12, Issue 7, pp 981–987

Stimulation and oxidative catalytic inactivation of thermolysin by copper•Cys-Gly-His-Lys

Original Paper

Abstract

[Cu2+•Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 μM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 μM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 μM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu2+–amino-terminal copper and nickel binding motif (λmax ∼ 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu2+ charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (ΔE = 92 mV) for the Cu3+/2+ redox couple obtained for [Cu2+•Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu2+•Lys-Gly-His-Lys]+ (0.84 V, ΔE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu2+•Lys-Gly-His-Lys]+ homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O2) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as kobs = 7.7 × 10−2 min−1, yielding a second-order rate constant of (7.7 ± 0.9) × 104 M−1 min−1. Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.

Keywords

Amino-terminal copper and nickel binding motif Thermolysin Drug design Catalytic inactivation Metallopeptides 

References

  1. 1.
    Endo S (1962) Hakko Kogaku Zasshi 40:346–353Google Scholar
  2. 2.
    Latt SA, Holmquist B, Vallee BL (1969) Biochem Biophys Res Commun 37:333–339PubMedCrossRefGoogle Scholar
  3. 3.
    Feder J, Garrett LR, Wildi BS (1971) Biochemistry 10:4552–4556PubMedCrossRefGoogle Scholar
  4. 4.
    Roques BP, Noble F, Dauge V, Fournie-Zaluski M-C, Beaumont A (1993) Pharmacol Rev 45:87–146PubMedGoogle Scholar
  5. 5.
    Cheng X-M, Ahn K, Haleen SJ (1997) Annu Rep Med Chem 32:61–70CrossRefGoogle Scholar
  6. 6.
    Wyvratt MJ, Patchett AA (1985) Med Res Rev 5:483–531PubMedCrossRefGoogle Scholar
  7. 7.
    Roques BP (1993) Biochem Soc Trans 21:678–685PubMedGoogle Scholar
  8. 8.
    Matthews BW (1988) Acc Chem Res 21:333–340CrossRefGoogle Scholar
  9. 9.
    Holmes MA, Matthews BW (1982) J Mol Biol 160:623–639PubMedCrossRefGoogle Scholar
  10. 10.
    Tiraboschi G, Jullian N, Thery V, Antonczak S, Fournie-Zaluski MC, Roques BP (1999) Protein Eng 12:141–149PubMedCrossRefGoogle Scholar
  11. 11.
    Bohacek R, De Lombaert S, McMartin C, Priestle J, Gruetter M (1996) J Am Chem Soc 118:8231–8249CrossRefGoogle Scholar
  12. 12.
    Fillion E, Gravel D (1996) Bioorg Med Chem Lett 6:2097–2102CrossRefGoogle Scholar
  13. 13.
    Gokhale NH, Cowan JA (2005) Chem Commun 5916–5918Google Scholar
  14. 14.
    Gokhale NH, Cowan JA (2006) J Biol Inorg Chem 11:937–947PubMedCrossRefGoogle Scholar
  15. 15.
    Cushman DW, Cheung HS, Sabo EF, Ondetti MA (1977) Biochemistry 16:5484–5491PubMedCrossRefGoogle Scholar
  16. 16.
    Gaucher JF, Selkti M, Tiraboschi G, Prange T, Roques BP, Tomas A, Fournie-Zaluski MC (1999) Biochemistry 38:12569–12576PubMedCrossRefGoogle Scholar
  17. 17.
    Gomez-Monterrey I, Beaumont A, Nemecek P, Roques BP, Fournie-Zaluski M-C (1994) J Med Chem 37:1865–1873PubMedCrossRefGoogle Scholar
  18. 18.
    Holmquist B, Vallee BL (1979) Proc Natl Acad Sci USA 76:6216–6220PubMedCrossRefGoogle Scholar
  19. 19.
    Peters K, Jahreis G, Kotters E-M (2001) J Enzyme Inhib 16:339–350PubMedGoogle Scholar
  20. 20.
    Roderick SL, Fournie-Zaluski MC, Roques BP, Matthews BW (1989) Biochemistry 28:1493–1497PubMedCrossRefGoogle Scholar
  21. 21.
    Harford C, Sarkar B (1997) Acc Chem Res 30:123–130CrossRefGoogle Scholar
  22. 22.
    McDonald MR, Scheper WM, Lee HD, Margerum DW (1995) Inorg Chem 34:229–237CrossRefGoogle Scholar
  23. 23.
    Tesfai TM, Green BJ, Margerum DW (2004) Inorg Chem 43:6726–6733PubMedCrossRefGoogle Scholar
  24. 24.
    Johnson GD, Ahn K (2000) Anal Biochem 286:112–118PubMedCrossRefGoogle Scholar
  25. 25.
    Segel I (1993) Enzyme kinetics: behavior and analysis of rapid equilibium and steady-state enzyme systems. Wiley Interscience, New YorkGoogle Scholar
  26. 26.
    Suh J (2003) Acc Chem Res 36:562–570PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2007

Authors and Affiliations

  • Nikhil H. Gokhale
    • 1
  • Seth Bradford
    • 1
  • J. A. Cowan
    • 1
  1. 1.Evans Laboratory of ChemistryOhio State UniversityColumbusUSA

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