Oxidative Inhibition of Human Soluble Catechol-O-methyltransferase

A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 °C. DTT also restored most of the activity that was lost upon incubation at 37 °C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role. A common polymorphism in the human gene for catechol-O-methyltransferase results in replacement of Val-108 by Met in the soluble form of the protein (s-COMT) and has been linked to breast cancer and neuropsychiatric disorders. The 108M and 108V variants are reported to differ in their thermal stability, with 108M COMT losing catalytic activity more rapidly. Because human s-COMT contains seven cysteine residues and includes CXXC and CXXS motifs that are associated with thiol-disulfide redox reactions, we examined the effects of reducing and oxidizing conditions on the enzyme. In the absence of a reductant 108M s-COMT lost activity more rapidly than 108V, whereas in the presence of 4 mm dithiothreitol (DTT) we found no significant differences in the stability of the two variants at 37 °C. DTT also restored most of the activity that was lost upon incubation at 37 °C in the absence of DTT. Mass spectrometry showed that cysteines 188 and 191 formed an intramolecular disulfide bond when s-COMT was incubated with oxidized glutathione, whereas cysteines 69, 95, 157, and 173 formed protein-glutathione adducts. Replacing Cys-95 by serine protected 108M s-COMT against inactivation in the absence of a reductant; C33S and Cys-188 mutations had little effect, and C69S was destabilizing. The sequences surrounding the reactive cysteine residues of human s-COMT and other proteins that form glutathione adducts at identified sites all include Pro and/or Gly and most include a hydrogen-bonding residue, suggesting that glutathiolation at conserved sites plays a physiologically important role. The enzyme catechol-O-methyltransferase (COMT, E.C. 2.1.1.6) modifies a variety of endogenous and exogenous catechol substrates by transferring a methyl group from S-adenosylmethionine (SAM) 1The abbreviations used are: SAM, S-adenosylmethionine; DTT, dithiothreitol; GSH and GSSG, reduced and oxidized glutathione; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; PSSG, protein-glutathione adduct; s-COMT and mb-COMT, the soluble and membrane-bound forms of catechol-O-methyltransferase, respectively; DTT, dithiothreitol.1The abbreviations used are: SAM, S-adenosylmethionine; DTT, dithiothreitol; GSH and GSSG, reduced and oxidized glutathione; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; PSSG, protein-glutathione adduct; s-COMT and mb-COMT, the soluble and membrane-bound forms of catechol-O-methyltransferase, respectively; DTT, dithiothreitol. to either the meta- or the para-hydroxyl group of the catechol ring. It plays important roles in the metabolism of catechol estrogens and the degradation of the catecholamine neurotransmitters dopamine and epinephrine. COMT is produced as both a soluble protein with 221 residues (s-COMT, 25 kDA) and a membrane-bound protein with an additional 50 residues at the N terminus (mb-COMT, 30 kDa) (1Huh M.M. Friedhoff A.J. J. Biol. Chem. 1979; 254: 299-308Abstract Full Text PDF PubMed Google Scholar). A single gene on human chromosome 22q11.2 encodes both proteins, but separate promoters initiate their expression (2Tenhunen J. Salminen M. Lundstrom K. Kiviluoto T. Savolainen R. Ulmanen I. Eur. J. Biochem. 1994; 223: 1049-1059Crossref PubMed Scopus (299) Google Scholar). The levels of expression of the two forms are tissue-specific; in the rat, s-COMT accounts for 95-99% of the enzyme in liver and most other tissues (3Tilgmann C. Kalkkinen N. FEBS Lett. 1990; 264: 95-99Crossref PubMed Scopus (35) Google Scholar), whereas mb-COMT is the major species in the adrenal medulla and some parts of the brain (4Ellingson T. Duddempudi S. Greenberg B.D. Hooper D. Eisenhofer G. J. Chromatogr. B. Biomed. Sci. Appl. 1999; 729: 347-353Crossref PubMed Scopus (38) Google Scholar). mb-COMT is found in the endoplasmic reticulum and the nuclear membrane (5Ulmanen I. Peranen J. Tenhunen J. Tilgmann C. Karhunen T. Panula P. Bernasconi L. Aubry J.P. Lundstrom K. Eur. J. Biochem. 1997; 243: 452-459Crossref PubMed Scopus (95) Google Scholar); s-COMT is found in the cytosol and nucleus (6Weisz J. Fritz-Wolz G. Gestl S. Clawson G.A. Creveling C.R. Liehr J.G. Dabbs D. Am. J. Pathol. 2000; 156: 1841-1848Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A common single-nucleotide polymorphism in the coding region of the human COMT gene results in substitution of methionine for valine at position 158 of mb-COMT and position 108 of s-COMT (7Weinshilboum R.M. Raymond F.A. Am. J. Med. Genet. 1977; 29: 125-135Google Scholar, 8Lachman H.M. Morrow B. Shprintzen R. Veit S. Parsia S.S. Faedda G. Goldberg R. Kucherlapati R. Papolos D.F. Am. J. Med. Genet. 1996; 67: 468-472Crossref PubMed Scopus (275) Google Scholar). In the general population of the United States, the frequencies of the Met/Met, Met/Val, and Val/Val genotypes are ∼0.17, 0.45, and 0.38, respectively (9Palmatier M.A. Kang A.M. Kidd K.K. Biol. Psychiatry. 1999; 46: 557-567Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). The COMT activity in erythrocyte lysates or liver biopsy samples from individuals with the Met/Met genotype is about one-fourth that in people with the Val/Val genotype, whereas heterozygous individuals have intermediate levels of activity. The 108M and 108V variants of s-COMT have similar kinetic properties (10Goodman J.E. Jensen L.T. He P. Yager J.D. Pharmacogenetics. 2002; 12: 517-528Crossref PubMed Scopus (61) Google Scholar, 11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar) but appear to differ in thermal stability (11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar, 12Vilbois F. Caspers P. Da Prada M. Lang G. Karrer C. Lahm H.-W. Cesura A.M. Eur. J. Biochem. 1994; 222: 377-386Crossref PubMed Scopus (29) Google Scholar, 13Dawling S. Roodi N. Mernaugh R.L. Wang X. Parl F.F. Cancer Res. 2001; 61: 6716-6722PubMed Google Scholar, 14Syvanen A.C. Tilgmann C. Rinne J. Ulmanen I. Pharmacogenetics. 1997; 7: 65-71Crossref PubMed Scopus (152) Google Scholar, 15Scanlon P.D. Raymond F.A. Weinshilboum R.A. Science. 1979; 203: 63-65Crossref PubMed Scopus (135) Google Scholar, 16Spielman R.S. Weinshilboum R.M. Am. J. Med. Genet. 1981; 10: 279-290Crossref PubMed Scopus (103) Google Scholar, 17Aksoy S. Klener J. Weinshilboum R.M. Pharmacogenetics. 1993; 3: 116-122Crossref PubMed Scopus (43) Google Scholar, 18Boudikova B. Szumlanski C. Maidak B. Weinshilboum R.M. Clin. Pharmacol. Ther. 1990; 48: 381-389Crossref PubMed Scopus (227) Google Scholar, 19Grossman M.H. Szumlanski C. Littrell J.B. Weinstein R. Weinshilboum R. Life Sci. 1992; 50: 473-480Crossref PubMed Scopus (26) Google Scholar). Lotta et al. (11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar) found that recombinant human 108M s-COMT lost 80% of its activity in 30 min at physiological temperature, whereas the 108V variant remained fully active. SAM protected the 108M enzyme against this loss of activity (11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar). Qualitatively similar differences between the apparent stabilities of the two variants have been seen in enzyme preparations from a variety of tissues (11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar, 12Vilbois F. Caspers P. Da Prada M. Lang G. Karrer C. Lahm H.-W. Cesura A.M. Eur. J. Biochem. 1994; 222: 377-386Crossref PubMed Scopus (29) Google Scholar, 13Dawling S. Roodi N. Mernaugh R.L. Wang X. Parl F.F. Cancer Res. 2001; 61: 6716-6722PubMed Google Scholar, 14Syvanen A.C. Tilgmann C. Rinne J. Ulmanen I. Pharmacogenetics. 1997; 7: 65-71Crossref PubMed Scopus (152) Google Scholar, 15Scanlon P.D. Raymond F.A. Weinshilboum R.A. Science. 1979; 203: 63-65Crossref PubMed Scopus (135) Google Scholar, 16Spielman R.S. Weinshilboum R.M. Am. J. Med. Genet. 1981; 10: 279-290Crossref PubMed Scopus (103) Google Scholar, 17Aksoy S. Klener J. Weinshilboum R.M. Pharmacogenetics. 1993; 3: 116-122Crossref PubMed Scopus (43) Google Scholar, 18Boudikova B. Szumlanski C. Maidak B. Weinshilboum R.M. Clin. Pharmacol. Ther. 1990; 48: 381-389Crossref PubMed Scopus (227) Google Scholar, 19Grossman M.H. Szumlanski C. Littrell J.B. Weinstein R. Weinshilboum R. Life Sci. 1992; 50: 473-480Crossref PubMed Scopus (26) Google Scholar). The 108/158M allele has been associated with increased risk of breast cancer (20Xie T. Ho S.L. Ramsden D. Mol. Pharmacol. 1999; 56: 31-38Crossref PubMed Scopus (280) Google Scholar, 21Tenhunen J. Heikkila P. Alanko A. Heinonen E. Akkila J. Ulmanen I. Cancer Lett. 1999; 144: 75-84Crossref PubMed Scopus (27) Google Scholar, 22Matsui A. Ikeda T. Enomoto K. Nakashima H. Omae K. Watanabe M. Hibi T. Kitajima M. Cancer Lett. 2000; 150: 23-31Crossref PubMed Scopus (51) Google Scholar, 23Mitrunen K. Jourenkova N. Kataja V. Eskelinen M. Kosma V.M. Benhamou S. Kang D. Vainio H. Uusitupa M. Hirvonen A. Cancer Epidemiol. Biomark. Prev. 2001; 10: 635-640PubMed Google Scholar) and a wide spectrum of mental disorders, including obsessive-compulsive disorder (24Karayiorgou M. Altemus M. Galke B.L. Goldman D. Murphy D.L. Ott J. Gogos J.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4572-4575Crossref PubMed Scopus (294) Google Scholar, 25Kirov G. Murphy K.C. Arranz M.J. Jones I. McCandles F. Kunugi H. Murray R.M. McGuffin P. Collier D.A. Owen M.J. Craddock N. Mol. Psychiatry. 1998; 3: 342-345Crossref PubMed Scopus (166) Google Scholar), ultra-rapid-cycling bipolar disorder (26Papolos D.F. Veit S. Faedda G.L. Saito T. Lachman H.M. Mol. Psychiatry. 1998; 3: 346-349Crossref PubMed Scopus (151) Google Scholar, 27Li T. Vallada H. Curtis D. Arranz M. Xu K. Cai G. Deng H. Liu J. Murray R. Liu X. Collier D.A. Pharmacogenetics. 1997; 7: 349-353Crossref PubMed Scopus (100) Google Scholar), certain manifestations of schizophrenia (28Strous R.D. Bark N. Parsia S.S. Volavka J. Lachman H.M. Psychiatry Res. 1997; 69: 71-77Crossref PubMed Scopus (225) Google Scholar, 29Kotler M. Barak P. Cohen H. Averbuch I.E. Grinshpoon A. Gritsenko I. Nemanov L. Ebstein R.P. Am. J. Med. Genet. 1999; 88: 628-633Crossref PubMed Scopus (168) Google Scholar, 30Nolan K.A. Volavka J. Czobor P. Cseh A. Lachman H. Saito T. Tiihonen J. Putkonen A. Hallikainen T. Kotilainen I. Rasanen P. Isohanni M. Jarvelin M.R. Karvonen M.K. Psychiatr. Genet. 2000; 10: 117-124Crossref PubMed Scopus (105) Google Scholar), anxiety (31Enoch M.A. Xu K. Ferro E. Harris C.R. Goldman D. Psychiatr. Genet. 2003; 13: 33-41Crossref PubMed Scopus (244) Google Scholar), and adult-onset alcoholism (32Tiihonen J. Hallikainen T. Lachman H. Saito T. Volavka J. Kauhanen J. Salonen J. Ryynanen O.P. Koulu M. Karvonen M.K. Pohjalainen T. Syvalahti E. Hietala J. Mol. Psychiatry. 1999; 4: 286-289Crossref PubMed Scopus (171) Google Scholar, 33Kauhanen J. Hallikainen T. Tuomainen T.P. Koulu M. Karvonen M.K. Salonen J.T. Tiihonen J. Alcohol. Clin. Exp. Res. 2000; 24: 135-139Crossref PubMed Google Scholar, 34Wang T. Franke P. Neidt H. Cichon S. Knapp M. Lichtermann D. Maier W. Propping P. Nothen M.M. Mol. Psychiatry. 2001; 6: 109-111Crossref PubMed Scopus (76) Google Scholar). It also has been linked to decreased responses of the μ-opioid system to pain (35Zubieta J.-K. Heitzeg M.M. Smith Y.R. Bueller J.A. Xu K. Xu Y. Koeppe R.A. Stohler C.S. Goldman D. Science. 2003; 299: 1240-1243Crossref PubMed Scopus (968) Google Scholar). A haplotype that combines the 108/158V allele with particular single-nucleotide polymorphisms in 2 non-coding regions of the gene is strongly associated with schizophrenia (36Shifman S. Bronstein M. Sternfeld M. Pisante-Shalom A. Lev-Lehman E. Weizman A. Reznik I. Spivak B. Grisaru N. Karp L. Schiffer R. Kotler M. Strous R.D. Swartz-Vanetik M. Knobler H.Y. Shinar E. Beckmann J.S. Yakir B. Risch N. Zak N.B. Darvasi A. Am. J. Hum. Genet. 2002; 71: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar), possibly because it results in decreased expression of the protein (37Bray N.J. Buckland P.R. Williams N.M. Williams H.J. Norton N. Owen M.J. O'Donovan M.C. Am. J. Hum. Genet. 2003; 73: 152-161Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The association of the 108/158M allele with mental dysfunction is particularly strong in patients with velocardiofacial syndrome, who lack the gene for COMT entirely on one copy of chromosome 22 and, thus, may be especially sensitive to the allele on the other copy (8Lachman H.M. Morrow B. Shprintzen R. Veit S. Parsia S.S. Faedda G. Goldberg R. Kucherlapati R. Papolos D.F. Am. J. Med. Genet. 1996; 67: 468-472Crossref PubMed Scopus (275) Google Scholar, 38Graf W.D. Unis A.S. Yates C.M. Sulzbacher S. Dinulos M.B. Jack R.M. Dugaw K.A. Paddock M.N. Parson W.W. Neurology. 2001; 57: 410-416Crossref PubMed Scopus (55) Google Scholar). No high-resolution structures of human COMT have been described. However, crystal structures of rat s-COMT in complex with SAM and catechol inhibitors are known (39Vidgren J. Svensson L. Lundstrom K. Nature. 1994; 364: 354-358Crossref Scopus (389) Google Scholar, 40Bonifácio M.J. Archer M. Rodrigues M.L. Matias P.M. Learmonth D.A. Carrondo M.A. Soares-Da-Silva P. Mol. Pharmacol. 2002; 62: 795-805Crossref PubMed Scopus (72) Google Scholar, 41Lerner C. Ruf A. Gramlich V. Masjost B. Zuercher G. Jakob-Roetne R. Borroni E. Diederich F. Angew. Chem. Int. Ed. Engl. 2001; 40: 4040-4042Crossref Scopus (59) Google Scholar), and the human protein probably is similar in structure because the amino acid sequences are 81% identical (see Figs. 1 and 2). The central structural motif, a seven-stranded β-sheet with helices on either side, is also characteristic of other SAM-dependent methyltransferases (42Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (400) Google Scholar). The SAM binding domain of human histamine methyltransferase, for example, is superimposable on rat s-COMT with a root mean square deviation of 2.9 Å over 156 Cα atoms (42Cheng X. Roberts R.J. Nucleic Acids Res. 2001; 29: 3784-3795Crossref PubMed Scopus (400) Google Scholar). The variable residue 108 of s-COMT is located in a loop between a helix and β-strand whose distal ends are close to the SAM binding site (Fig. 1). The rat protein has a leucine residue at position 108 and no known polymorphism at that location. Remarkably, however, histamine-N-methyltransferase has a common single-nucleotide polymorphism (105Thr/Ile) at almost exactly the same position. In that case, the two variants of the enzyme have somewhat different kinetic properties but similar thermal stabilities (43Horton J.R. Sawada K. Nishibori M. Zhang X. Cheng X. Structure. 2001; 9: 837-849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Fig. 2Amino acid sequences of human, pig, rat, mouse, frog, fish, and protozoan s-COMT as aligned by ClustalW. Cysteine residues are indicated by bold C. The total number of cysteines in each protein is indicated, and CXXS and CXXC motifs are labeled.View Large Image Figure ViewerDownload (PPT) There has been little discussion of structural differences that might explain the different stabilities or activities of the 108V and 108M variants of human s-COMT. Whether the inactivation of the 108M enzyme reflects gross unfolding or a more subtle change in the structure is unknown. Weinshilboum and co-workers (17Aksoy S. Klener J. Weinshilboum R.M. Pharmacogenetics. 1993; 3: 116-122Crossref PubMed Scopus (43) Google Scholar, 19Grossman M.H. Szumlanski C. Littrell J.B. Weinstein R. Weinshilboum R. Life Sci. 1992; 50: 473-480Crossref PubMed Scopus (26) Google Scholar) examined the proteins by polyacrylamide gel electrophoresis, isoelectric focusing, immune fixation, and photoaffinity labeling, and found no significant differences between the Met and Val variants. However, a possible clue comes from early observations that rat s-COMT loses activity on storage but can be reactivated by the reducing agent dithiothreitol (DTT) (44Assicot M. Bohuoh C. Eur. J. Biochem. 1970; 12: 490-495Crossref PubMed Scopus (50) Google Scholar). Of the 20 common amino acids, cysteine is the most sensitive to oxidation; the thiol groups of exposed cysteines readily oxidize to form intra- or intermolecular disulfide bridges. Cysteine is one of the least abundant amino acids in proteins and also has the highest sequence conservation. Rat s-COMT contains four cysteines (residues 33, 69, 157, and 188), whereas human s-COMT has seven (33, 69, 95, 157, 173, 188, and 191). Two of the three additional cysteines in the human protein (Cys-95 and Cys-173) are close to the active site and may contribute to or modulate substrate binding (Fig. 1). The third (Cys-191) along with Cys-188 forms a CXXC motif, which is found in thioredoxins, glutaredoxins, DsbA, and protein disulfide isomerase, proteins that undergo reversible thiol/disulfide oxidation/reduction reactions (45Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 46Woycechowsky K.J. Raines R.T. Curr. Opin. Chem. Biol. 2000; 4: 533-539Crossref PubMed Scopus (99) Google Scholar, 47Arner E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1976) Google Scholar, 48Fomenko D.E. Gladyshev V.N. Biochemistry. 2003; 42: 11214-11225Crossref PubMed Scopus (125) Google Scholar) (Fig. 2). In addition, Cys-69 is part of a CXXS motif that is found in many proteins with thiol/disulfide oxido-reductase activities (49Fomenko D.E. Gladyshev V.N. Protein Sci. 2002; 11: 2285-2296Crossref PubMed Scopus (62) Google Scholar). Fomenko and Gladyshev (49Fomenko D.E. Gladyshev V.N. Protein Sci. 2002; 11: 2285-2296Crossref PubMed Scopus (62) Google Scholar) suggest that the local secondary structure and hydrogen bonding with the serine hydroxyl group makes cysteines in this motif particularly prone to oxidation. Vilbois et al. (12Vilbois F. Caspers P. Da Prada M. Lang G. Karrer C. Lahm H.-W. Cesura A.M. Eur. J. Biochem. 1994; 222: 377-386Crossref PubMed Scopus (29) Google Scholar) showed that cysteines 33, 69, 95, and 173 of human s-COMT react readily with the thiol reagent 5-iodoacetamide fluorescein and that this treatment inactivates the enzyme. They further showed that SAM and MgCl2 decrease the reaction with cysteines 69 and 95 and partially protect the enzyme against inactivation by the thiol reagent, suggesting that cysteine 69, 95, or both are essential for catalytic activity (12Vilbois F. Caspers P. Da Prada M. Lang G. Karrer C. Lahm H.-W. Cesura A.M. Eur. J. Biochem. 1994; 222: 377-386Crossref PubMed Scopus (29) Google Scholar). However, no intramolecular disulfide linkages were detected via mass spectroscopy in those studies. With the above observations in mind it occurred to us that the reported differences between the thermal stabilities of the 108V and 108M variants of human s-COMT could reflect differences in susceptibility to oxidation or in the effects of oxidation on catalytic activity. The presence of two redox-associated motifs further suggested that cysteine oxidation and/or glutathiolation might be part of a mechanism for regulating COMT activity or, in the case of glutathiolation, for protecting the protein under conditions of oxidative stress. Although many investigators have included DTT or β-mercaptoethanol in assays for COMT activity, there have been no published studies of the effects of oxidants or reductants on the difference between the thermal stabilities or activities of the Val and Met variants. To our knowledge, mutations of Cys residues in human COMT also have not been investigated. However, Männistö et al. (50Männistö P.T. Ulmanen I. Lundström K. Taskinen J. Tenhunen J. Tilgmann C. Kaakkola S. Prog. Drug Res. 1992; 39: 291-350PubMed Google Scholar) report without details that a C33A mutation destroys the activity of rat s-COMT; the C69A, C157A, and C191A mutant enzymes were said to be active (as mentioned above, rat s-COMT does not have cysteines at positions 95, 173, and 191). In the present work we have compared the enzymatic activities of the 108V and 108M human s-COMT variants at physiological temperature in the presence or absence of DTT. Additionally, we have studied the effects of C33S, C69S, C95S, and C188S mutations on the enzymatic activity and stability of the 108M and 108V variants of recombinant human s-COMT. We also have looked for changes in the redox states of the cysteine residues when the 108M and 108V enzymes are incubated under oxidizing or reducing conditions and have examined the reactivation of the oxidized enzymes by DTT. Chemicals—S-(5′-Adenosyl)-l-methionine (SAM), 4′-hydroxy-3′-methoxyacetophenone, 1,4-dithio-l-threitol, l-glutathione, isopropyl-1-thio-β-d-galactopyranoside, DTT and phenylmethylsulfonyl fluoride were obtained from Sigma-Aldrich, 4′-methoxy-3′-hydroxyacetophenone was from Taizhou Dongdong Pharmachem (China), and 3′,4′-dihydroxyacetophenone was from Oakwood Products, Inc. Cloning, Expression, and Purification—Dr. David Eaton and Helen Smith (Department of Environmental Health, University of Washington) provided a c-DNA clone of 108V human s-COMT in the Novagen pET22b(+) vector that contains a C-terminal histidine tag. Starting with the 108V clone, we mutated Thr-39 to Ala to agree with the NCBI sequence for human s-COMT (NP_009294.1) and then introduced the V108M mutation followed by C33S, C69S, C95S, and C188S mutations for both the 108V and 108M variants. All the mutations were made using the QuikChange site-directed mutagenesis kit (Stratagene). DNA from each strain was sequenced to verify the mutation and ensure that no unintended changes had occurred. Recombinant s-COMT was expressed in Escherichia coli BL21*(DE3) cells (Stratagene). Cell cultures with an absorbance of ∼0.65 at 595 nm were induced with 500 μm isopropyl-1-thio-β-d-galactopyranoside and grown for 6 h at 37 °C. Cultures were centrifuged at 4000 × g for 15 min at 4 °C, and the pellets were re-suspended and disrupted by sonication (50% duty, 80% power for 10 min) in 100 mm Tris-HCl, pH 8, 300 mm NaCl, 1 mm EDTA, 10% glycerol (v/v), 5 mm β-mercaptoethanol, 5 mm MgCl2, and 10 μm phenylmethylsulfonyl fluoride. The cell extract was centrifuged at 12,000 × g for 20 min at 4 °C to pellet any insoluble material. The resulting supernatant was passed through a filter with 0.22-μm pores (Millipore) and applied to a 4-ml column of Talon Metal Affinity resin (Clontech). Nonspecifically bound proteins were cleared using 30 ml of wash buffer (100 mm Tris-HCl, pH 7.5, 200 mm NaCl, 5 mm MgCl2, 5 mm β-mercaptoethanol) followed by 30 ml of wash buffer with 7 mm imidazole. COMT then was eluted using two 6-ml volumes of elution buffer (wash buffer plus 100 mm imidazole) and pooled into 6 ml of ice-cold storage buffer (100 mm Tris-HCl, pH 7.5, 200 mm NaCl, 10% glycerol (v/v), 5 mm DTT, 5 mm MgCl2). This procedure yielded up to 15 mg (∼0.6 μmol) of COMT per liter of culture. The concentrated protein was dialyzed overnight at 4 °C in 2-liter storage buffer and stored at -20 °C. To remove DTT and mercaptoethanol, enzyme stocks were thawed, exchanged into buffer lacking a reducing agent (50 mm Tris HCl, pH 7.5, 1.5 mm MgCl2) using Quick Spin protein columns (Roche Applied Science), and returned to -20°C for storage. Protein concentration was determined by the Bradford assay (51Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar) or, in a few cases, ultraviolet absorbance (52Gill S.C. von Hipple P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5034) Google Scholar). The latter gave somewhat lower enzyme concentrations and, thus, higher specific activities. COMT Enzymatic Activity—The assay mixture contained 1 μm recombinant COMT, 50 mm Tris-HCl, pH 7.5, 1.5 mm MgCl2, and except where indicated otherwise, 4 mm DTT in a total volume of 250 μl. The reaction was initiated by adding 20 μm dihydroxyacetophenone (Km ∼ 10 μm (53Borchardt R.T. Anal. Biochem. 1974; 58: 382-389Crossref PubMed Scopus (27) Google Scholar, 54Lautala P. Ulmanen I. Taskinen J. Mol. Pharmacol. 2001; 59: 393-402Crossref PubMed Scopus (95) Google Scholar)) and 200 μm SAM (Km ∼ 50 μm (11Lotta T. Vidgren J. Tilgmann C. Ullmanen I. Melen K. Biochemistry. 1995; 34: 4202-4210Crossref PubMed Scopus (992) Google Scholar)), allowed to proceed for 15 or 30 min at either 22 or 37 °C, and terminated by the addition of 150 μl of saturated NaCl and 250 μl of ethyl acetate and vortexing. After the mixture had separated into aqueous and organic phases, 150 μl of the organic phase was removed for analysis by gas chromatography. The methylated products (4′-hydroxy-3′-methoxyacetophenone and 4′-methoxy-3′-hydroxyacetophenone) were separated as shown in Fig. 3 in an Agilent Technologies 5890B gas chromatography apparatus with a fused silica capillary column (30-m × 0.25-mm × 0.25-μm film thickness, Agilent Technologies HP5), a 95% methyl-, 5% phenyl-silicone stationary phase, and a mass spectrometer (Agilent Technologies 5970B). 1 μl of sample was injected in the splitless mode, and helium at a starting pressure of 15 p.s.i. was used as the carrier gas in constant-flow mode. The oven temperature was increased from 70 to 250 °C over a total run time of 15 min. The solvent delay was 3 min. The retention times of the two products were separated by ∼1 min. The products were identified by comparison with standards and by mass spectrometry. The mass spectrometer was operated in the electron-impact mode using single-ion-monitoring at m/z = 166. A standard curve relating concentration to the integrated peak area of the gas chromatogram was produced for each product. Enzyme activities are expressed as μm methylated products (3′-hydroxy-4′methoxyacetophenone plus 3′-methoxy-4′-hydroxyacetophenone) formed per unit time (30 or 15 min, as indicated) per μm protein, and the values given are the mean and S.D. of the mean of three measurements. The meta/para methylation ratio typically was between 1.3 and 1.6. Reaction with Glutathione, Proteolysis, and Matrix-assisted Laser-desorption Ionization Time-of-flight (MALDI-TOF Mass Spectrometry—Human s-COMT (∼20 μm) was incubated for 5 h at 4 °C in 10 mm Tris HCl, pH 7.5, 2 mm MgCl2, and 1 mm NaCl with or without 7 mm DTT or 5 mm oxidized glutathione (GSSG). 10 μl of 5 mm iodoacetamide, an alkylating agent, was then added to 10 μl of protein samples. After 45 min in the dark at room temperature, the protein was digested with 200 ng of sequencing grade trypsin (Promega) for 2 h at 37 °C. The digestion was stopped by the addition of acetic acid to a final concentration of 1%. The proteolyzed material was analyzed with a MALDI-TOF mass spectrometer (Voyager DE-Pro, Applied Biosystems). A 1-μl fraction of proteolyzed sample was mixed with 2.0 μl of matrix solution (0.1% trifluoroacetic acid, 50% acetonitrile saturated with α-cyano-4-hydroxycinammic acid), and 1.0 μl of the mixture was spotted onto a stainless steel target and allowed to dry. Positive-ion spectra were collected in the reflector mode. The mass accuracy of fragment ions was ±1 Da. A standard peptide mixture was used for external calibration. Immunoblotting—After treatment with oxidized glutathione as described above, human s-COMT (20-40 μg) was submitted to SDS-PAGE under non-reducing conditions and then transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences). The protein was exposed to an IgG2a mouse monoclonal antibody that recognizes glutathione-protein disulfide adducts (Virogen). Adherent antibody was detected by reaction with horseradish peroxidase-conjugated goat anti-mouse secondary antibody and enhanced chemiluminescence detection (Pierce). Bioinformatics and Homology Modeling—Amino acid sequences were aligned using ClustalW (www.ebi.ac.uk/clustalw). The CYSPRED algorithm (55Fiser A. Simon I. Bioinformatics. 2000; 16: 251-256Crossref PubMed Scopus (79) Google Scholar) was used to pre