Rate Constants and Equilibrium Constants for Thiol-Disulfide Interchange Reactions Involving Oxidized Glutathione

Journal of the American Chemical Society

Volumn 102, Issue 6, 1980, Pages 2011-2026

  Szajewski, Richard P ^a   Whitesides, George M ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 33847087446     PISSN: 00027863     EISSN: 15205126     Source Type: Journal    
DOI: 10.1021/ja00526a042     Document Type: Article

References (48)

1. Parts of this research were supported by NIH (GM 26543, 24025, and CA 09112CT).
2. NIH Postdoctoral Fellow, 1976-1978 (Fellowship A105337).
3. M. Friedman, “The Chemistry and Biochemistry of the Sulfhydryl Group”, 1st ed.t Pergamon Press, Elmsford, N.V., 1973; P. C. Jocelyn, “Biochemistry of the SH Group”, Academic Press, New York, 1972; Yu. M. Tor-chinskii, “Sulfhydryl and Disulfide Groups of Proteins”, Plenum Press, New York, 1974; A. L. Flaharty in “The Chemistry of the Thiol Group”, S. Patai, Ed., Wiley, New York, 1974, p 589.
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8. Hydrogen peroxide is generated during most oxidations of thiols by dioxygen: P. P. Trotto, L. M. Pinkus, and A. Meister, J. Biol. Chem., 249,1915 (1974); M. Costa, L. Pecci, B. Pensa, and C. Cannella, Biochem. Blophys. Res. Commun., 78, 596 (1977).
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10. E. Bernt and H. U. Bergmeyer in “Methods of Enzymatic Analysis”, Vol. 4, 2nd ed., H. U. Bergmeyer, Ed., Academic Press, New York, 1974, p 1643.
11. W. E. Knox in “The Enzymes”, Vol. 2, 2nd ed., P. Boyer, Ed., Academic Press, New York, 1960, p 253; E. Boyland and L. F. Chasseaud, Adv. Enzymol., 32, 173 (1969); A. Meister in “The Enzymes”, Vol. 10,3rd ed., P. Boyer, Ed., Academic Press, New York, 1974, p 671; N. S. Kosower and E. M. Kosower in “Free Radicals in Biology”, Vol. II, W. A. Pryor, Ed., Academic Press, New York, 1976, Chapter II; I. M. Arias and W. B. Jakoby, “Glutathione: Metabolism and Function”, Raven Press, New York, 1976.
12. G. M. Whitesides, J. Lilburn, and R. P. Szajewski, J. Org. Chem., 42, 332 (1977).
13. J. M. Wilson, R. J. Bayer, and D. J. Hupe, J. Am. Chem. Soc., 99, 7922 (1977); C. E. Grimshaw, R. L. Whistler, and W. W. Cleland, J. Org. Chem., 101, 1521 (1979); M. Shipton and K. Brocklehurst, Biochem. J., 171, 385 (1978); K. Brocklehurst and G. Little, J. Org. Chem., 128, 471 (1972). The value of βnu0 0.45 has been reported for the reaction of amines with disulfides: H. Al-Rawl, K. A. Stacy, R. H. Weatherhead, and A. William, J. Chem. Soc., Pbrkin Trans. 2, 663(1978).
14. The substrate for glyoxalase I is the hemithioacetal formed between glutathione-SH and an a-keto aldehyde. See E. E. Cliffe and S. C. Waley, Biochem. J., 79, 475 (1961); M. V. Kester and S. J. Norton, Biochim. Biophys. Acta, 391, 212 (1975); L.-P. B. Han, L. M. Davison, and D. L. Vander Jagt, J. Am. Chem. Soc., 445, 486 (1976); D. L. Vander Jagt, E. Daub, J. A. Krohn, and L.-P. B. Han, Biochemistry, 14, 3669 (1975). For a discussion of the magnitudes of equilibrium constants between GSH and aldehydes, see M. S. Kanchuger and L. D. Byers, J. Am. Chem. Soc., 101, 3005 (1979).
15. D. M. E. Reuben and T. C. Bruice, J. Am. Chem. Soc., 98, 114 (1976). Robenstein [D. L. Robenstein, J. Am. Chem. Soc., 95, 2797 (1973)] reports microscopic pKas of 8.93 (amino group protonated) and 9.08 (amino group unprotonated) based on NMR chemical shifts, and Jung [G. Jung, Eur. J. Biochem., 24, 438 (1972)] reports pKa = 9.2. M. S. Kanchuger and L. D. Byers, J. Am. Chem. Soc., 101, 3005 (1979), Indicated pKa = 9.1 by alkylation. The origin of the differences between these values is not entirely clear. In any event, the estimate of Bruice (pKa = 8.7) is compatible with our data, and is used throughout this paper.
16. The maximum specific activity reported for this enzyme is ~103 fimo\ of product mg-1 min~1. Using a molecular weight of 48 00014 for GX-I, this value gives a turnover number of ~5 X 105 mol of product min-1 (mol of enzyme)-1 as the maximum value of (k3). Typically, kinetic runs were performed using enzyme with specific activity of ~200 U mg-1.
17. N. Kharasch, “Organic Sulphur Compounds”, Vol. I. Pergamon Press, Elmsford, N.Y., 1961; A. Fava, I. Illiceto, and E. Camera, J. Am. Chem. Soc., 79, 833 (1957); L. Eldjarn and A. Pihl, J. Biol. Chem., 225, 499 (1957).
18. H. U. Bergmeyer in ref 10, Vol. 1, p 95 ff.
19. H. R. Mahler and E. H. Cordes, “Biological Chemistry”, Harper and Row, New York, 1966, p 237 ff.
20. R. H. Weaver and H. A. Lardy, J. Biol. Chem., 236, 313 (1961).
21. The values of /c,°bsd in this work are comparable to rates (1.9-33 M-1 min-1) observed for the formation of GSSG from GSH and GSSR at pH 7.0; U. Weber, P. Hartter, and L. Flohe, Hoppe-Seyier's Z. Physiol. Chem., 351, 1389 (1970). The values of k, in this work are also comparable to those found by J.-R. Garel, FEBS Lett., 79, 135 (1977), for the reduction of a cystine S-S bond in ribonuclease A of 3.0 X 102 M~1 min-1 at pH 8.5.
22. T. E. Creighton, J. Mol. Biol., 96, 767 (1975). Several corrections were applied to Creighton's data before they were incorporated into Figure 5. First, the p/Ca values are those used consistently throughout this paper, and do not always match those used by Creighton. Second, Creighton's rate constants are calculated on the basis of the appearance of oxidized DTT, and ours on the appearance of reduced thiol by reaction of a disulfide with a monothiol. Creighton's rate constants were multiplied by a factor of 4 to make them compatible with ours: a factor of 2 to account for the difference in the rates of appearance of oxidized DTT and reduced thiol (2d[DTT0X]/df = d[RS~]/df), and a second factor of 2 to account for the presence of two symmetry-equivalent thiol groups in DTT.
23. A. A. Frost and R. G. Pearson, “Kinetics and Mechanisms”, 2nd ed., Wiley, New York, 1961.
24. S. S. Hall, A. M. Doweyko, and F. Jordan, J. Am. Chem. Soc., 100, 5934 (1978).
25. The value of ftuc given in ref 12 was calculated from the rate constants incorrectly, and is in error.
26. The data of Hupe et al. also show some small sensitivity to the particular data included in the analysis. Two sets of experiments were carried out in obtaining rate constants for reductions’ with aryl thiols: one used the reducing agent in excess, one had Ellman's reagent in excess. Least-squares analysis of the first set of data yielded βnuoaryl = 0.48 (r2 = 0.96); analysis of the second set of data yielded βnuca'yl = 0.41 (i2 = 1.0); analysis of both together yielded ftucaryl = 0.44 (r2 = 0.97). Inclusion of a point for methyl mercaptoacetate in Hupe's analysis changes ftucalkyl to 0.43 (r2 = 0.91).
27. Although the data for alkyl thiols in these studies are in good agreement, there is a small but real difference in the data for the aryl thiols. The similarity in the alkyl thiol rate constants suggests that no consistent experimental difference accounts for this difference. All of our data were obtained using the same procedure, although their experimental precision was lower for the more rapidly reacting thiols. Hupe et al. used slightly different procedures for alkyl and aryl thiols. Either this difference in procedure, or differences in the compositions of solutions (especially ionic strength and buffer composition), might contribute to the consistent factor of ca. 2 which separates these sets of rate constants.
28. We note that the values of ftuc derived from these two independent investigations are, in fact, experimentally indistinguishable (β.uc yl = 0.4112 vs. O.4313) if the datum for methyl mercaptoacetate is included in the calculation of the latter point. Hupe et al. point out that this compound shows deviations in analyses of rate constants derived from both thioesters and disulfides, and exclude it on that basis.
29. C. D. Johnson, Chem. Rev., 75, 755 (1975).
30. H. A. Smith, G. Doughty, and G. Gorin, J. Org. Chem., 29, 1484 (1964).
31. G. Dalman, J. McDermed, and G. Gorin, J. Org. Chem., 29, 1480 (1964).
32. D. R. Sanadi, M. Langley, and R. L. Searls, J. Biol. Chem., 234, 178 (1959), report E0’ for the reduction of lipoamide to be ’0.294 V at pH 7.1, 22 °C, vs. the standard hydrogen electrode. This corresponds to a value of ’0.288 V at pH 7.0, 30 °C. V. Massey, Biochim. Biophys. Acta, 37, 314 (1960), reports a value of ’0.288 V vs. standard hydrogen electrode at pH 7.0, 30 °C. We have repeated the equilibrations described by Dandi et al. and obtained the same value for this potential. These potentials were all obtained by equilibration against an NAD+/NADH couple. We have used a value of Erf ’ ’0.30 V at pH 7.0, 30.0 °C, vs. standard hydrogen electrode for this couple: K. Burton and T. H. Wilson, Biochem. J., 54, 86 (1953). A Erf values and equilibrium constants were interconverted using eq 33; K27 = (lipooxXNADH)/(lipored)(NADH+) = 0.0858 at pH 7.0, 30 °C. K27 (and Knad063'1) are thus pH dependent but K27[H+]_1 (and KNADobM [H+]-1) are pH independent.
33. UV spectra of solutions obtained by mixing BAL and Ellman's reagent showed only those features attributable to Ellman's anion and to acyclic S-S bonds (Ellman's reagent). However, consideration of the trends observed in the UV of various disulfides34 (acyclic, emax 480 at X 250 nm; 1,2-dithiepane, fmax 444 at Xmax 258 nm; 1,2-dithiane, emax 290 at Xmax 290 nm; 1,2-dithiolane, fmax 147 at X 330) suggest that the for a four-membered cyclic disulfide might be vanishingly small. Raman spectra of an identical mixture of BAL and Ellman's reagent show, in addition to the expected absorptions, a new band at 510 cm-1. This position is consistent with there being no special strain in the S-S bond and is substantially different from the v 500 cm-1 predicted for a 1,2-dithietane.35 Thus, these spectral data are compatible with formation of either a cyclic dimer or higher oligomer or a polymer on oxidation of BAL. The isolation of a dithietane has not been reported, but stable disulfide-containing rings containing seven or more members are well established: A. E. Asato and R. E. Moore, Tetrahedron Lett., 4841 (1973); G. H. Wahl, J. Bordner, D. N. Harpp, and J. G. Gleason, J. Chem. Soc. Chem. Commun., 985 (1972); T. C. Owen, J. M. Fayach, and J. S. Chen, J. Org. Chem., 38, 937 (1973).
34. J. A. Barltrap, P. M. Hayes, and M. Calvin, J. Am. Chem. Soc., 76, 4348 (1954).
35. H. E. Van Wart and H. A. Scheraga, J. Phys. Chem., 80, 1812, 1823 (1976).
36. We assume in doing these calculations that the pKa of HSR-SSR’ equals that of HSRSH (see Discussion section in text). The calculated values of Kgssgs and KQSS0SH include a statistical factor of 2 whenever a dithiol reducing agent is involved.
37. J. P. Snyder and L. Carlsen, J. Am. Chem. Soc., 99, 2931 (1977).
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40. R. P. Bell, “The Proton in Chemistry”, 2nd ed., Cornell University Press, Ithaca, N.Y., 1973; W. P. Jencks, “Catalysis in Chemistry and Enzymology”, McGraw-Hill, New York, 1969.
41. The effective molarity of thiol in intramolecular attack on the carbonyl moiety of p-nitrophenyl AA2-mercaptophenyl)-/V-methylcarbamate is 1.4 X 10= M: cf. T. H. Fife, J. E. C. Hutchins, and M. S. Wang, J. Am. Chem. Soc., 97, 5878 (1975). For a general discussion of intramolecular effects see M. I. Page, Angew. Chem., Int. Ed. Engl., 16, 449 (1977).
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43. S. Oae, “Organic Chemistry of Sulfur”, Plenum Press, New York, 1977, Chapter 7; B. Meyer, Chem. Rev., 76, 367 (1976).
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46. K. Gawehn and H. U. Bergmeyer in ref 10, Vol. 3, p 1496.
47. H. U. Bergmeyer, K. Gawehn, and M. Grassl in ref 10, Vol. 1, p 469.
48. N. F. Chamberlain and J. J. R. Reed in “The Analytical Chemistry of Sulfur and Its Compounds”, Part III, “Nuclear Magnetic Resonance Data of Sulfur Compounds”, J. H. Karchmer, Ed., Wiley, New York, 1971.