AAPS PharmSci

, Volume 2, Issue 1, pp 68–77

Comparison of methods for analyzing kinetic data from mechanism-based enzyme inactivation: Application to nitric oxide synthase

Article

Abstract

The goals of this study were (1) to investigate the performance of 2 classical methods of kinetic analysis when applied to data from enzyme systems in which mechanism-based inactivation and enzyme degradation are present, and (2) to develop and validate a nonlinear method of kinetic data analysis that may perform better under these situations. A composite equation was derived to link various parameters that govern the kinetics of mechanism-based inactivation, viz., enzyme activity, inhibitor-binding affinity (K1), inactivation rate (Kinact), and enzyme degradation (kdeg). The relative accuracy and precision of parameter estimation by the Dixon and Kitz-Wilson methods and a new nonlinear method were evaluated by computer simulation. The behavior of these methods of analysis were validated experimentally, using the nitric oxide synthase enzyme, both in purified form and as expressed in murine macrophage cell cultures. We showed that the Dixon method, as expected, could not provide accurate estimates of K1 in the presence of either enzyme inactivation or instability. The Kitz-Wilson method could provide accurate estimates of these parameters; however, the precisions of these estimates were poorer than those obtained using the nonlinear method of analysis. We conclude that the nonlinear approach is superior to classical methods of data analysis for enzyme inhibitor kinetics, based on better efficiency, accuracy, and precision.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Cheng Y, Prusoff WH. Relationship between the inhibition constant KI and the concentration of inhibitor which causes 50 per cent inhibition 150 of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–3108.PubMedCrossRefGoogle Scholar
  2. 2.
    Furfine ES, Harmon MF, Paith JE, Knowles, RG, Salter M, Kiff, RJ, et al. Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. J Biol Chem. 1994;269:26677–26683.PubMedGoogle Scholar
  3. 3.
    Garvey EP, Oplinger JA, Tanoury GJ, Sherman PA, Fowler M, Marshall S, et al. Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. J Biol Chem 1994;269:26669–26676.PubMedGoogle Scholar
  4. 4.
    Garvey EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJ, et al. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997;272:4959–4963.PubMedCrossRefGoogle Scholar
  5. 5.
    Bertz RJ, Granneman GR. Use of in vitro and in vivo data to estimate the likelihood of metabolic pharmacokinetic interactions. Clin Pharmacokinet. 1997;32:210–258.PubMedCrossRefGoogle Scholar
  6. 6.
    Ghosh SS, Said-Nejad O, Roestamadji J, Mobashery S. The first mechanism-based inactivators for angiotensin-converting enzyme. J Med Chem. 1992;35:4175–4179.PubMedCrossRefGoogle Scholar
  7. 7.
    Kalgutkar AS, Castagnoli Jr N. Synthesis of novel MPTP analogs as potential monoamine oxidase B MAO-B inhibitors. J Med Chem 1992;35:4165–4174.PubMedCrossRefGoogle Scholar
  8. 8.
    Wade ML, Voelkel NF, Fitzpatrick FA. “Suicide” inactivation of prostaglandin 12 synthase: characterization of mechanism-based inactivation with isolated enzyme and endothelial cells. Arch Biochem Biophys. 1995;321:453–458.PubMedCrossRefGoogle Scholar
  9. 9.
    Wolff DJ, Gauld DS, Neulander MJ, Southan G. Inactivation of nitric oxide synthase by substituted aminoguanidines and aminoisothioureas. J Pharmacol Exp Ther. 1997;283:265–273.PubMedGoogle Scholar
  10. 10.
    Marks GS, McCluskey SA, Mackie JE, Riddick DS, James CA. Disruption of hepatic heme biosynthesis after interaction of xenobiotics with cytochrome P-450. FASEB J. 1988;2:2774–2783.PubMedGoogle Scholar
  11. 11.
    Bondon A, Macdonald TL, Harris TM, Guengerich FP. Oxidation of cycloalkylamines by cytochrome P-450. Mechanism-based inactivation, adduct formation, ring expansion, and nitrone formation. J Biol Chem 1989;264:1988–1997.PubMedGoogle Scholar
  12. 12.
    Rando RR. Mechanism-based enzyme inactivators. Pharmacol Rev. 1984;36:111–142.PubMedGoogle Scholar
  13. 13.
    Rando RR. Chemistry and enzymology of keat inhibitors. Science. 1974;85:320–324.CrossRefGoogle Scholar
  14. 14.
    Palfreyman MG, Bey P, Sjoerdsma A. Enzymeactivated mechanism-based inhibitors. Essays Biochem. 1987;23:28–81.PubMedGoogle Scholar
  15. 15.
    Chen PF, Tsai AL, Wu KK. Cysteine 99 of endothelial nitric oxide synthase NOS-III is critical for tetrahydrobiopterin-dependent NOS-III stability and activity. Biochem Biophys Res Comm. 1995;215:1119–1129.PubMedCrossRefGoogle Scholar
  16. 16.
    Kitz R, Wilson IB. Esters of methanesulfonic acid as irreversible inhibitors of Acetylcholinesterase. J Biol Chem. 1962;237:3245–3249.PubMedGoogle Scholar
  17. 17.
    Dixon M. The determination of enzyme inhibitor constants. Biochem J. 1952;55:170–171.Google Scholar
  18. 18.
    Silverman R. Mechanism-based Enzyme Inactivation: Chemistry and Enzymology. Boca Raton, FL. CRC Press, 1988.Google Scholar
  19. 19.
    Reif DW, McCreedy SA. N-nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation toward the three nitric oxide synthases. Arch. Biochem Biophys. 1995;320:170–176.PubMedCrossRefGoogle Scholar
  20. 20.
    Olken NM, Marletta MA. NG-allyl-and NG-cyclopropyl-L-arginine: two novel inhibitors of macrophage nitric oxide synthase. J Med Chem. 1992;35:1137–1144.PubMedCrossRefGoogle Scholar
  21. 21.
    Olken NM, Rusche KM, Richards MK, Marletta MA. Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine. Biochem Biophys Res Comm 1991;177:828–833.PubMedCrossRefGoogle Scholar
  22. 22.
    Boje KM, Fung HL. Endothelial nitric oxide generating enzymes in the bovine aorta: subcellular location and metabolic characterization. J Pharmacol Exp Ther 1990;253:20–26.PubMedGoogle Scholar
  23. 23.
    Motulsky HJ, Ransnas LA. Fitting curves to data using nonlinear regression a practical and nonmathematical review. FASEB J. 1987;1:365–374.PubMedGoogle Scholar
  24. 24.
    Olken NM, Marletta MA. NG-methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase. Biochemistry. 1993;32:9677–9685.PubMedCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2002

Authors and Affiliations

  1. 1.Department of Pharmaceutical TechnologyKobe Pharmaceutical UniversityKobeJapan
  2. 2.Department of Pharmaceutics, School of PharmacyUniversity at Buffalo, State University of New YorkBuffalo

Personalised recommendations