Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 May 15;220(1):51–55. doi: 10.1042/bj2200051

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.

M X Sliwkowski, H E Swaisgood, D A Clare, H R Horton
PMCID: PMC1153593  PMID: 6743273

Abstract

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

Full text

PDF
51

Selected References

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

  1. Ashkar S., Binkley F., Jones D. P. Resolution of a renal sulfhydryl (glutathione) oxidase from gamma-glutamyltransferase. FEBS Lett. 1981 Feb 23;124(2):166–172. doi: 10.1016/0014-5793(81)80128-7. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Clare D. A., Blakistone B. A., Swaisgood H. E., Horton H. R. Sulfhydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase. Arch Biochem Biophys. 1981 Oct 1;211(1):44–47. doi: 10.1016/0003-9861(81)90427-6. [DOI] [PubMed] [Google Scholar]
  4. Cohen H. J., Fridovich I. Hepatic sulfite oxidase. Purification and properties. J Biol Chem. 1971 Jan 25;246(2):359–366. [PubMed] [Google Scholar]
  5. Cornish-Bowden A., Eisenthal R. Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta. 1978 Mar 14;523(1):268–272. doi: 10.1016/0005-2744(78)90030-x. [DOI] [PubMed] [Google Scholar]
  6. Eisenthal R., Cornish-Bowden A. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J. 1974 Jun;139(3):715–720. doi: 10.1042/bj1390715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. FLAMM W. G., CRANDALL D. I. Purification of mammalian homogentisate oxidase and evidence for the existence of ferrous mercaptans in the active center. J Biol Chem. 1963 Jan;238:389–396. [PubMed] [Google Scholar]
  8. Fridovich I. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J Biol Chem. 1970 Aug 25;245(16):4053–4057. [PubMed] [Google Scholar]
  9. Janolino V. G., Sliwkowski M. X., Swaisgood H. E., Horton H. R. Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A. Arch Biochem Biophys. 1978 Nov;191(1):269–277. doi: 10.1016/0003-9861(78)90089-9. [DOI] [PubMed] [Google Scholar]
  10. Janolino V. G., Swaisgood H. E. Isolation and characterization of sulfhydryl oxidase from bovine milk. J Biol Chem. 1975 Apr 10;250(7):2532–2538. [PubMed] [Google Scholar]
  11. Kobashi K. Catalytic oxidation of sulfhydryl groups by o-phenanthroline copper complex. Biochim Biophys Acta. 1968 May;158(2):239–245. doi: 10.1016/0304-4165(68)90136-0. [DOI] [PubMed] [Google Scholar]
  12. McCord J. M., Fridovich I. The reduction of cytochrome c by milk xanthine oxidase. J Biol Chem. 1968 Nov 10;243(21):5753–5760. [PubMed] [Google Scholar]
  13. Misra H. P. Generation of superoxide free radical during the autoxidation of thiols. J Biol Chem. 1974 Apr 10;249(7):2151–2155. [PubMed] [Google Scholar]
  14. Ogasawara N., Gander J. E., Henderson L. M. Purification and properties of 3-hydroxyanthranilate oxygenase from beef kidney. J Biol Chem. 1966 Feb 10;241(3):613–619. [PubMed] [Google Scholar]
  15. Ostrowski M. C., Kistler W. S., Williams-Ashman H. G. A flavoprotein responsible for the intense sulfhydryl oxidase activity of rat seminal vesicle secretion. Biochem Biophys Res Commun. 1979 Mar 15;87(1):171–176. doi: 10.1016/0006-291x(79)91662-0. [DOI] [PubMed] [Google Scholar]
  16. Robinson J., Cooper J. M. Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Anal Biochem. 1970 Feb;33(2):390–399. doi: 10.1016/0003-2697(70)90310-6. [DOI] [PubMed] [Google Scholar]
  17. Schmelzer C. H., Swaisgood H. E., Horton H. R. Resolution of renal sulfhydryl oxidase from gamma-glutamyltransferase by covalent chromatography on cysteinylsuccinamidopropyl-glass. Biochem Biophys Res Commun. 1982 Jul 16;107(1):196–201. doi: 10.1016/0006-291x(82)91688-6. [DOI] [PubMed] [Google Scholar]
  18. Sliwkowski M. B., Sliwkowski M. X., Swisgood H. E., Horton H. R. Nonidentity of sulfhydryl oxidase and gama-glutamyltransferase in bovine milk. Arch Biochem Biophys. 1981 Oct 15;211(2):731–737. doi: 10.1016/0003-9861(81)90509-9. [DOI] [PubMed] [Google Scholar]
  19. Sliwkowski M. X., Sliwkowski M. B., Horton H. R., Swaisgood H. E. Resolution of sulphydryl oxidase from gamma-glutamyltransferase in bovine milk by covalent chromatography on cysteinylsuccinamidopropyl-glass. Biochem J. 1983 Mar 1;209(3):731–739. doi: 10.1042/bj2090731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Takamori K., Thorpe J. M., Goldsmith L. A. Skin sulfhydryl oxidase. Purification and some properties. Biochim Biophys Acta. 1980 Oct;615(2):309–323. doi: 10.1016/0005-2744(80)90499-4. [DOI] [PubMed] [Google Scholar]
  21. WILKINSON G. N. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. doi: 10.1042/bj0800324. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES