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• W2085457861 abstract "Monoamine oxidases (MAO) A and B are ∼60-kDa outer mitochondrial membrane flavoenzymes catalyzing the degradation of neurotransmitters and xenobiotic arylalkyl amines. Despite 70% identity of their amino acid sequences, both enzymes exhibit strikingly different properties when exposed to thiol-modifying reagents. Human MAO A and MAO B each contain 9 cysteine residues (7 in conserved sequence locations). MAO A is inactivated by N-ethylmaleimide (NEM) much faster (τ½ = ∼3 min) than MAO B (τ½ = ∼8 h). These differences in thiol reactivities are also demonstrated by monitoring the NEM modification stoichiometries by electrospray mass spectrometry. Inactivation of either enzyme with acetylenic inhibitors results in alterations of their thiol reactivities. Cys5 and Cys266 were identified as the only residues modified by biotin-derivatized NEM in clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively. The x-ray structure of MAO B (Binda, C., Newton-Vinson, P., Hubalek, F., Edmondson, D. E., and Mattevi, A. (2002) Nat. Struct. Biol. 9, 22–26) shows that Cys5 is located on the surface of the molecule opposite to the membrane-binding region. Cys266 in MAO A is predicted to be located in the same region of the molecule. These thiol residues are also modified by biotin-derivatized NEM in the mitochondrial membrane-bound MAO A and MAO B. This study shows that the MAO A structure is “more flexible” than that of MAO B and that clorgyline and pargyline inactivation of MAO A and B, respectively, increases the structural stability of both enzymes. No evidence is found for the presence of disulfide bonds in either enzyme, contrary to a previous suggestion. Monoamine oxidases (MAO) A and B are ∼60-kDa outer mitochondrial membrane flavoenzymes catalyzing the degradation of neurotransmitters and xenobiotic arylalkyl amines. Despite 70% identity of their amino acid sequences, both enzymes exhibit strikingly different properties when exposed to thiol-modifying reagents. Human MAO A and MAO B each contain 9 cysteine residues (7 in conserved sequence locations). MAO A is inactivated by N-ethylmaleimide (NEM) much faster (τ½ = ∼3 min) than MAO B (τ½ = ∼8 h). These differences in thiol reactivities are also demonstrated by monitoring the NEM modification stoichiometries by electrospray mass spectrometry. Inactivation of either enzyme with acetylenic inhibitors results in alterations of their thiol reactivities. Cys5 and Cys266 were identified as the only residues modified by biotin-derivatized NEM in clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively. The x-ray structure of MAO B (Binda, C., Newton-Vinson, P., Hubalek, F., Edmondson, D. E., and Mattevi, A. (2002) Nat. Struct. Biol. 9, 22–26) shows that Cys5 is located on the surface of the molecule opposite to the membrane-binding region. Cys266 in MAO A is predicted to be located in the same region of the molecule. These thiol residues are also modified by biotin-derivatized NEM in the mitochondrial membrane-bound MAO A and MAO B. This study shows that the MAO A structure is “more flexible” than that of MAO B and that clorgyline and pargyline inactivation of MAO A and B, respectively, increases the structural stability of both enzymes. No evidence is found for the presence of disulfide bonds in either enzyme, contrary to a previous suggestion. Monoamine oxidases (MAO) 1The abbreviation used are: MAO, monoamine oxidase(s); Biotinyl-NEM, polyethylene oxide maleimide-activated biotin; ESI, electrospray ionization; MS, mass spectrometry; NEM, N-ethylmaleimide; HPLC, high performance liquid chromatography; SELDI, surface enhanced laser desorption ionization; LC, liquid chromatography.1The abbreviation used are: MAO, monoamine oxidase(s); Biotinyl-NEM, polyethylene oxide maleimide-activated biotin; ESI, electrospray ionization; MS, mass spectrometry; NEM, N-ethylmaleimide; HPLC, high performance liquid chromatography; SELDI, surface enhanced laser desorption ionization; LC, liquid chromatography. A and B are outer mitochondrial membrane enzymes whose function is to catalyze the oxidative deamination of neurotransmitters (serotonin, dopamine, and norepinephrin), dietary amines (phenylethylamine), and arylalkylamine-containing drugs used in numerous therapies (1Weyler W. Hsu Y.P. Breakefield X.O. Pharmacol. Ther. 1990; 47: 391-417Crossref PubMed Scopus (290) Google Scholar). Human enzymes are pharmacological targets for antidepressants and neuro-protective agents. Although MAO A and MAO B have similar catalytic activities, they differ in substrate specificities and tissue distribution (1Weyler W. Hsu Y.P. Breakefield X.O. Pharmacol. Ther. 1990; 47: 391-417Crossref PubMed Scopus (290) Google Scholar, 2Rodriguez M.J. Saura J. Billett E.E. Finch C.C. Mahy N. Cell Tissue Res. 2001; 304: 215-220Crossref PubMed Scopus (54) Google Scholar). Exploitation of subtle differences in MAO A and MAO B structures is desirable to establish isoform-specific drugs. Human MAO A and MAO B are encoded by separate genes and share a sequence homology of ∼70% (3Bach A.W. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (697) Google Scholar). Activity of either enzyme is sensitive to thiol-modifying reagents (4Weyler W. Salach J.I. J. Biol. Chem. 1985; 260: 13199-13207Abstract Full Text PDF PubMed Google Scholar, 5Barron E.S.G. Singer T.P. J. Biol. Chem. 1945; 157: 221-240Abstract Full Text PDF Google Scholar), and cysteine residues have been implicated in the catalytic mechanism of MAO based on the results of site-directed mutagenesis (6Wu H.F. Chen K. Shih J.C. Mol. Pharmacol. 1993; 43: 888-893PubMed Google Scholar, 7Cesura A.M. Gottowik J. Lahm H.W. Lang G. Imhof R. Malherbe P. Rothlisberger U. Da Prada M. Eur. J. Biochem. 1996; 236: 996-1002Crossref PubMed Scopus (34) Google Scholar), thiol titration (8Gomes B. Kloepfer H.G. Oi S. Yasunobu K.T. Biochim. Biophys. Acta. 1976; 438: 347-357Crossref PubMed Scopus (14) Google Scholar), and inhibitor binding (9Zhong B. Silverman R.B. J. Am. Chem. Soc. 1997; 119: 6690-6691Crossref Scopus (42) Google Scholar) experiments. Human MAO A and MAO B each contain 9 cysteine residues with 7 of them in highly conserved positions (Scheme 1; A165/B156, A201/B192, A306/B297, A321/B312, A374/B365, A398/B389, and A406/B397) (3Bach A.W. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (697) Google Scholar). 2Numbering for both enzymes refers to the gene-predicted amino acid sequences. On the protein level, the initial Met residue of MAO B is removed.2Numbering for both enzymes refers to the gene-predicted amino acid sequences. On the protein level, the initial Met residue of MAO B is removed. In each enzyme, one conserved cysteine residue (Cys406 in MAO A and Cys397 in MAO B) is linked in a thioether bond to the 8α-methylene of FAD (10Weyler W. Biochem. J. 1989; 260: 725-729Crossref PubMed Scopus (34) Google Scholar, 11Kearney E.B. Salach J.I. Walker W.H. Seng R.L. Kenney W. Zeszotek E. Singer T.P. Eur. J. Biochem. 1971; 24: 321-327Crossref PubMed Scopus (193) Google Scholar), therefore leaving 8 cysteine residues either as free thiols or in disulfide bonds. Gomes et al. (8Gomes B. Kloepfer H.G. Oi S. Yasunobu K.T. Biochim. Biophys. Acta. 1976; 438: 347-357Crossref PubMed Scopus (14) Google Scholar) reported that 2 cysteine residues in bovine liver MAO B are protected from reaction with thiol probes by substrates, concluding that these 2 residues are located to the active site and essential for enzyme catalytic activity. The proximity of Cys365 to the catalytic site in bovine MAO B was proposed by Zhong and Silverman (9Zhong B. Silverman R.B. J. Am. Chem. Soc. 1997; 119: 6690-6691Crossref Scopus (42) Google Scholar) by demonstrating this residue to be modified on inhibition of bovine liver MAO B with N-cyclopropyl-α-methylbenzylamine. Mutagenesis experiments by Wu et al. (6Wu H.F. Chen K. Shih J.C. Mol. Pharmacol. 1993; 43: 888-893PubMed Google Scholar) have shown Ser mutations of Cys374 and Cys406 to abolish MAO A activity and Ser mutations of Cys156, Cys365, and Cys397 to abolish MAO B activity. Mutations of the other cysteine residues were shown not to alter catalytic activities of either enzyme (6Wu H.F. Chen K. Shih J.C. Mol. Pharmacol. 1993; 43: 888-893PubMed Google Scholar). Cesura et al. (7Cesura A.M. Gottowik J. Lahm H.W. Lang G. Imhof R. Malherbe P. Rothlisberger U. Da Prada M. Eur. J. Biochem. 1996; 236: 996-1002Crossref PubMed Scopus (34) Google Scholar) found that MAO B activity is decreased significantly when Cys365 is mutated to Ala. The recently solved crystal structure of MAO B (12Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (531) Google Scholar) reveals that Cys365 and Cys156, previously suggested to be involved in catalytic mechanism (6Wu H.F. Chen K. Shih J.C. Mol. Pharmacol. 1993; 43: 888-893PubMed Google Scholar, 7Cesura A.M. Gottowik J. Lahm H.W. Lang G. Imhof R. Malherbe P. Rothlisberger U. Da Prada M. Eur. J. Biochem. 1996; 236: 996-1002Crossref PubMed Scopus (34) Google Scholar, 9Zhong B. Silverman R.B. J. Am. Chem. Soc. 1997; 119: 6690-6691Crossref Scopus (42) Google Scholar), are located on the surface of the molecule. A recent study (13Sablin S.O. Ramsay R.R. J. Biol. Chem. 1998; 273: 14074-14076Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) on the stoichiometry of reduction of bovine MAO B or of recombinant human liver MAO A by sodium dithionite led to the conclusion that each enzyme contains a redox active disulfide group, which was suggested to function catalytically in shuttling reducing equivalent between the amine substrate and the FAD. This suggestion has been questioned because anaerobic titration of either enzyme requires stoichiometric levels of substrate to reduce the enzyme-bound flavin coenzyme (14Li M. Hubalek F. Newton-Vinson P. Edmondson D.E. Prot. Expres. Purif. 2002; 24: 152-162Crossref PubMed Scopus (80) Google Scholar, 15Newton-Vinson P. Hubalek F. Edmondson D.E. Prot. Expres. Purif. 2000; 20: 334-345Crossref PubMed Scopus (104) Google Scholar). To address these uncertainties that exist in the literature regarding the role of thiol groups in MAO A and MAO B structure and function, a detailed study was conducted using purified enzyme preparations. The availability of large scale quantities of purified recombinant, fully functional human MAO A (14Li M. Hubalek F. Newton-Vinson P. Edmondson D.E. Prot. Expres. Purif. 2002; 24: 152-162Crossref PubMed Scopus (80) Google Scholar) and MAO B (15Newton-Vinson P. Hubalek F. Edmondson D.E. Prot. Expres. Purif. 2000; 20: 334-345Crossref PubMed Scopus (104) Google Scholar) in our laboratory facilitated this approach. Both enzymes were examined for the presence of disulfide bonds using mass spectrometry by analyzing the thiol-modified intact enzymes prior to and after any disulfide bond reduction. Cysteine reactivities in functional purified enzymes are also compared before and after inactivation by clorgyline or pargyline, their respective acetylenic inactivators (16Youdim M.B.H. Collins G.G.S. Sandler M. Jones A.B.B. Pare C.M.B. Nicholson W.J. Nature. 1972; 236: 225-228Crossref PubMed Scopus (83) Google Scholar, 17Fowler C.J. Mantle T.J. Tipton K.F. Biochem. Pharmacol. 1982; 31: 3555-3561Crossref PubMed Scopus (108) Google Scholar, 18Chuang H.Y. Patek D.R. Hellerman L. J. Biol. Chem. 1974; 249: 2381-2384Abstract Full Text PDF PubMed Google Scholar). The level of resolution achieved by electrospray ionization quadrupole mass spectrometry allows for monitoring of the distribution of reacted thiol groups in the enzymes (19Whitelegge J.P. le Coutre J. Lee J.C. Engel C.K. Prive G.G. Faull K.F. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10695-10698Crossref PubMed Scopus (106) Google Scholar). In addition, the positions of reactive cysteine residues were determined. The results reported in this manuscript demonstrate that neither MAO A nor MAO B contains any disulfide bonds and that despite the differences in cysteine reactivities between the two enzymes in their native states, cysteine reactivities of enzymes inactivated with clorgyline or pargyline are similar and also provide further documentation for the suggested binding mode of MAO B to the outer mitochondrial membrane. Materials—All buffers and reagents were purchased in the highest grade available from Sigma unless specified otherwise. β-Octylglucopyranoside was purchased from Labscientific Inc. (Livingston, NJ), N-ethylmaleimide (NEM), and EZ-Link polyethylene oxide maleimideactivated biotin (Biotinyl-NEM) were from Pierce, formic acid was from EM Science (Gibbstown, NJ), and N,N-dimethylformamide was from Honeywell Burdick and Jackson (Muskegon, MI). Enzyme Preparation—Both human recombinant MAO A and MAO B were expressed in Pichia pastoris and purified as described previously (14Li M. Hubalek F. Newton-Vinson P. Edmondson D.E. Prot. Expres. Purif. 2002; 24: 152-162Crossref PubMed Scopus (80) Google Scholar, 15Newton-Vinson P. Hubalek F. Edmondson D.E. Prot. Expres. Purif. 2000; 20: 334-345Crossref PubMed Scopus (104) Google Scholar); MAO B was kindly provided by Dr. P. Newton-Vinson and MAO A by Min Li in our laboratory. Prior to each experiment, the enzymes (1–2 mg) were desalted using a G-25 (fine) Sephadex column (1 × 20 cm) in 50 mm potassium phosphate buffer, pH 7.5, containing 0.8% (w/v) β-octylglucopyranoside (Buffer A). Disulfide Bond Analysis—To identify the number of cysteine residues reacting with NEM in the fully reduced form of the enzyme, MAO A (10 μm) was first incubated for 30 min at 25 °C in Buffer A containing 2 m guanidine HCl and 4 mm reduced dithiothreitol; NEM was then added to a final concentration of 11 mm. One hour following the addition of NEM, the reaction was quenched with 70 μl of formic acid/N,N-dimethylformamide/water (23%/70%/7% v/v/v, Solution B), desalted by microbore reversed-phase HPLC, and analyzed by ESI-MS (14Li M. Hubalek F. Newton-Vinson P. Edmondson D.E. Prot. Expres. Purif. 2002; 24: 152-162Crossref PubMed Scopus (80) Google Scholar, 15Newton-Vinson P. Hubalek F. Edmondson D.E. Prot. Expres. Purif. 2000; 20: 334-345Crossref PubMed Scopus (104) Google Scholar). The number of cysteine residues reacting with NEM in the native (unreduced, as isolated) form of the enzyme was identified by incubating MAO A (10 μm) with 100 μm NEM at 25 °C for 2 h in Buffer A containing 2 m guanidine HCl. The reaction was quenched with 70 μl of Solution B and analyzed by microbore HPLC and ESI-MS. The same experiments were performed with human recombinant MAO B. N-Ethylmaleimide or Biotinyl-NEM Modification—Aliquots of 8 mm solutions of NEM or Biotinyl-NEM were added to the enzyme (10 μm in Buffer A) to a final concentration of 800 μm (MAO-to-NEM ratio of 1:80), and the resulting solutions were incubated at either 0 °C or 25 °C. At specified times, 5 μl aliquots of the reaction mixtures were analyzed for catalytic activity using the kynuramine assay for MAO A (14Li M. Hubalek F. Newton-Vinson P. Edmondson D.E. Prot. Expres. Purif. 2002; 24: 152-162Crossref PubMed Scopus (80) Google Scholar) or the benzylamine assay for MAO B (15Newton-Vinson P. Hubalek F. Edmondson D.E. Prot. Expres. Purif. 2000; 20: 334-345Crossref PubMed Scopus (104) Google Scholar), and 100-μl aliquots of the reaction mixtures were quenched with 70 μl of Solution B and stored at –20 °C pending HPLC and MS analyses. Tryptic Digestion—Dried HPLC-purified Biotinyl-NEM-modified MAO was redissolved in 1 μl of 80% formic acid and immediately diluted with 10 μl of 50% aqueous isopropanol and 90 μl of 0.1 m ammonium bicarbonate. The pH of this solution was adjusted to ∼8 by adding 5% ammonium hydroxide. Aliquots of trypsin (1 μg; Promega, Madison, WI, sequencing grade) were added to a final trypsin-to-MAO ratio of ∼1:30 (mol/mol), and the mixture was incubated in the dark at 37 °C for 24 h. Streptavidin Affinity Chromatography—To isolate Biotinyl-NEM modified peptides, the tryptic digests were applied to a streptavidin affinity cartridge (Applied Biosystems) according to the manufacturer's protocol. The resulting eluates were analyzed by ESI-LC-MS using a Zorbax SB-C18 (0.5 × 150 mm; Agilent Technologies) capillary HPLC column or by SELDI-MS. Samples for SELDI-MS analysis in 50% isopropanol, 30% formic acid were loaded onto protein chips (H4 protein chips; Ciphergen Biosystems, Fremont, CA) and washed with 0.1% aqueous formic acid prior to sinapinic acid (matrix) addition. (These conditions were found to increase the recovery of hydrophobic peptides.) In some experiments, an internal standard biotinylated peptide (ALSEGC(Biotinyl-NEM)TPYDIN; molecular mass = 1826.6 Da, maleamic acid form; molecular mass = 1808.6 Da, maleimide form) was added (100 pmol) to the MAO digest prior to streptavidin chromatography. Biotinyl-NEM Modification of MAO A and MAO B in Intact Mitochondria—Intact mitochondria were isolated from P. pastoris overexpressing either MAO A or MAO B as described earlier (20Urban P. Andersen J.K. Hsu H.P. Pompon D. FEBS Let. 1991; 286: 142-146Crossref PubMed Scopus (31) Google Scholar). Intact mitochondria containing 1 unit of either MAO A or MAO B in Hepes buffer (10 mm Hepes, pH 7.4, containing 1 mm phenylmethylsulfonyl fluoride) were incubated with 20-fold molar excess of clorgyline and pargyline, respectively, on ice for 20 min, following which the suspensions were centrifuged at 20,000 × g for 10 min. After washing the pellet with Hepes buffer, the mitochondria were resuspended in Hepes buffer and incubated with Biotinyl-NEM (final concentration, 1 mm; ∼1/100 mol/mol MAO/Biotinyl-NEM) for 15 h at 4 °C. The excess Biotinyl-NEM was removed by centrifugation (20,000 × g for 10 min). The mitochondria were than resuspended in SDS-PAGE sample buffer, and mitochondrial proteins were separated using SDS-PAGE (7.5% gel) and stained with Coomassie Brilliant Blue. MAO-containing bands were cut out of the gel and digested with trypsin (21Cole A.M. Liao H.I. Stuchlik O. Tilan J. Pohl J. Ganz T. J. Immunol. 2002; 169: 6985-6991Crossref PubMed Scopus (161) Google Scholar) prior to streptavidin affinity chromatography and MS analyses (as described above). For Western blot analysis of biotinylated proteins, the gels were electro-blotted onto a nitrocellulose membrane, and the membrane was incubated with streptavidin conjugated to alkaline phosphatase (Pierce) and developed using a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma). Disulfide Bond Analysis—MAO A and MAO B each contain 9 cysteine residues (Scheme 1) (3Bach A.W. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (697) Google Scholar). One cysteine residue (A407/B397) is covalently attached to FAD (10Weyler W. Biochem. J. 1989; 260: 725-729Crossref PubMed Scopus (34) Google Scholar, 11Kearney E.B. Salach J.I. Walker W.H. Seng R.L. Kenney W. Zeszotek E. Singer T.P. Eur. J. Biochem. 1971; 24: 321-327Crossref PubMed Scopus (193) Google Scholar), leaving 8 cysteine residues as free thiol groups or involved in disulfide bonds. When purified recombinant MAO A and MAO B were incubated separately with NEM following guanidine hydrochloride denaturation with or without dithiothreitol reduction, ESI-MS of either MAO preparation showed an increase in mass consistent with reaction of 8 mol of NEM/mol of enzyme (Fig. 1). The fact that the same increase in mass is independent of whether the enzyme preparations were pretreated with dithiothreitol demonstrates that all 8 cysteine residues in either purified MAO preparation are available for NEM modification. Based on these results, we conclude that there are no disulfide bonds present in either MAO A or MAO B. Kinetics of NEM Modification—MAO A and MAO B inactivation with sulfhydryl-reactive reagents proceeds with different kinetic time courses (Table I). MAO A is inactivated by an 80-fold molar excess of NEM at 25 °C with a τ½ of ∼2 min, whereas MAO B is inactivated with a τ½ of ∼8 h under the same conditions. At 0 °C, MAO A is inactivated with a τ½ of ∼3 h, and no inactivation of MAO B is observed (τ½ > 100 h). MAO B is inactivated at a faster rate (τ½ = ∼3 h) when an 800-fold excess of NEM was used at 25 °C. All of the enzyme inactivations exhibit pseudo-first-order kinetic behavior, with the exception of MAO A inactivation at 25 °C, which is biphasic, in agreement with previous reports (13Sablin S.O. Ramsay R.R. J. Biol. Chem. 1998; 273: 14074-14076Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Table IHalf-times for MAO A and MAO B inactivation with NEM and Biotinyl NEMτ½ MAO Aτ½ MAO B25 °C0 °C25 °C0 °CminhhhUntreateda10 μm MAO A or MAO B in 50 mm potassium phosphate, pH 7.5, containing 0.8% octyl glucoside were used in all experiments.309030>1000.8 mm NEM (80-fold excess)2bFollows biphasic kinetics.38>1008 mm NEM (800-fold excess)NDcND, not determined.ND3ND0.8 mm Biotinyl-NEM (80-fold excess)<514>100>100a 10 μm MAO A or MAO B in 50 mm potassium phosphate, pH 7.5, containing 0.8% octyl glucoside were used in all experiments.b Follows biphasic kinetics.c ND, not determined. Open table in a new tab The ability of quadrupole ESI-MS to resolve mass differences corresponding to the addition of single NEM (∼125 Da) allowed the monitoring of the time course of the reaction. MAO A reacts with >7 NEM molecules during 1 h of incubation at 25 °C (Fig. 2A), whereas clorgyline-inactivated MAO A reacts with 5 NEM molecules over a 2-h period (Fig. 2B). Two clusters of modified MAO B are observed after 24 h of incubation with NEM at 25 °C, a low stoichiometry cluster (1–4 NEM groups added) and a high stoichiometry cluster (8–10 NEM groups added) (Fig. 2C). These results suggest that once MAO B is modified with up to 4 NEM groups, it is structurally altered to promote further modification, resulting in the fully S-alkylated (8 NEM groups) and “overmodified” enzyme (>8 NEM groups). Pargyline inactivation of MAO B prevents the formation of the high stoichiometry cluster, suggesting that pargyline binding to MAO B prevents the structural changes necessary for modification of all of its thiol groups (Fig. 2D). Similar results to these reported above for MAO A were observed when MAO B was incubated with an 800-fold excess of NEM at 25 °C for 5 h (data not shown). These results suggest that clorgyline and pargyline inactivations of MAO A and MAO B, respectively, provide additional structural stability for each enzyme. This is demonstrated by modification of three more cysteine residues in MAO A as compared with clorgyline-inactivated MAO A (Fig. 2, A and B) and, similarly, by modification of five more cysteine residues in MAO B as compared with pargyline-inactivated MAO B (Fig. 2, C and D). In some cases, residues other than cysteine are modified by NEM, resulting in instances where MAO B is modified by more than 8 NEM groups (Fig. 2C), especially when large excess of NEM over MAO B is used (data not shown). This finding is not surprising because NEM has previously been reported to react (albeit at slower rates) with residues such as histidine, lysine, and tyrosine when used in large excess (22Gehring H. Christen P. Methods Enzymol. 1983; 91: 392-396Crossref PubMed Scopus (4) Google Scholar). Under all experimental conditions used, cysteine residues in MAO A are more reactive than cysteine residues in MAO B. Kinetics of Biotinyl-NEM Modification—Although NEM is suitable for detailed kinetic studies, we found it difficult to directly analyze NEM-modified cysteine-containing tryptic peptides of MAO A and MAO B because of their hydrophobicity. Therefore we used water-soluble EZ-link Biotinyl-NEM (Scheme 2) to aid in the identification of specific cysteine reactive sites. Biotinyl-NEM was used to modify reactive cysteine residues because it facilitates rapid and selective purification of the labeled peptides using streptavidin chromatography (see below). The inactivation half-times for MAO A are τ½ = ∼5 min at 25 °C and τ½ = ∼14 h at 0 °C when the enzyme was incubated with an 80-fold excess of Biotinyl-NEM. Under the same conditions, MAO B is very slow to inactivate at either temperature (τ½ > 100 h). Multiple cysteine residues (up to 7) in MAO A react with Biotinyl-NEM after 24 h of incubation at 0 °C, whereas only a single cysteine residue in clorgyline-inactivated MAO A reacts with Biotinyl-NEM under the same reaction conditions (Fig. 3). A single cysteine residue in MAO B and pargyline-inactivated MAO B reacts with Biotinyl-NEM under the same conditions (Fig. 3). Despite the large differences in cysteine reactivities between MAO A and MAO B, both the clorgyline- and pargyline-inactivated enzymes react with only a single Biotinyl-NEM group, suggesting that clorgyline binding to MAO A stabilizes the MAO A structure. Biotinyl-NEM reacted with fewer cysteine residues than NEM under identical conditions. This is most likely due to its size and hydrophilic properties, which diminish its access to the protein core.Fig. 3Influence of clorgyline/pargyline inactivation on Biotinyl-NEM modification of MAO A and MAO B. Deconvoluted ESI-MS spectra of MAO A (left panel) and MAO B (right panel) after incubation with an 80-fold excess of Biotinyl-NEM at 0 °C for 24 h are shown. Clorgyline inactivation had a major effect on MAO A cysteine reactivities (bottom left panel), resulting in a significantly slower rate of Biotinyl-NEM modification as compared with the inhibitor free enzyme (top left panel). Pargyline inactivation had little effect on the reactivities of MAO B cysteine residues (bottom right panel) as compared with the inhibitor free enzyme (top right panel). The peaks are labeled with the number of Biotinyl-NEM groups bound to the enzyme.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of Modified Cysteine Residues—To determine the locations of the single Biotinyl-NEM-modified cysteine residues in clorgyline-inactivated MAO A and pargyline-inactivated MAO B (Fig. 3), biotinylated tryptic peptides were enriched by streptavidin affinity chromatography and analyzed by either LC-ESI-MS or SELDI-MS. The use of SELDI-MS, which allowed for the deposition of large sample volumes onto the SELDI sample plate and subsequent sample desalting, has proved essential for the analyses. Peptide 240–267, containing Cys266, was identified as the only peptide modified by Biotinyl-NEM in clorgyline-inactivated MAO A (Fig. 4). A relatively high background is observed in this spectrum, which is due to the presence of unmodified MAO A tryptic peptides that were retained throughout the purification, likely because of their hydrophobicity. Upon the first analysis of peptides from a singly biotinylated MAO B by SELDI-MS, no biotinylated peptide was identified, although the internal standard was readily observed. Only after detailed analysis of minor species present in the mass spectrum, peptide 5–21, containing Cys5, was identified as the only peptide modified by Biotinyl-NEM in pargyline-inactivated MAO B (Fig. 4). None of the other minor species in the spectrum correspond to a biotinylated MAO B peptide. The low recovery of this peptide is not surprising given its very nonpolar nature (CDVVVVGGGISGMAAAK). In addition, because of hydrolysis of the maleimide ring (23Gregory J.D. J. Am. Chem. Soc. 1955; 77: 3922-3923Crossref Scopus (268) Google Scholar), two forms of this peptide were detected resulting in even lower recovery (maleimide form, molecular mass = 2060 Da, ∼60%; maleamic acid form, molecular mass = 2078 Da, ∼40%, Fig. 4). In contrast, the biotinylated peptide of MAO A was almost completely recovered in its maleamic acid form. 3This phenomenon could be explained by the presence of histidine residues within the sequence of the MAO A peptide that could facilitate ring hydrolysis by acting as a general base. Similar findings, when maleimide-modified peptides from the same protein showed different degrees of the ring hydrolysis, were observed earlier (F. Hubálek, unpublished data). Structural Equivalence of Cys5 (MAO B) and Cys266 (MAO A)—Although Cys5 in MAO B and Cys266 in MAO A are not aligned in the amino acid sequences of the two MAOs, they show similar reactivities toward Biotinyl-NEM. The recently solved x-ray structure of MAO B (12Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (531) Google Scholar) reveals that Cys5 (MAO B) is located on the surface of the molecule opposite to the membrane-binding region. Cys266 (MAO A) aligns with Ala257 (MAO B) when the MAO A and MAO B sequences are aligned using a Clustal X algorithm using the Blosum 30 matrix (24Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55635) Google Scholar). Ala257 is located in the same region of the molecule as Cys5 (Fig. 5), suggesting that Cys5 in MAO B and Cys266 in MAO A are structurally equivalent. The slower rate of modification of Cys266 in MAO A relative to Cys5 in MAO B (Fig. 3) could be explained by steric hindrance of Cys266 by Phe14 in MAO A (Fig. 5). In contrast, the thiol group of Cys5 in MAO B is quite accessible to solvent. Analysis of MAO A and MAO B in Intact Mitochondria—To determine whether the conclusions from the above experiments performed with detergent-solubilized enzyme" @default.
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• W2085457861 title "Structural Comparison of Human Monoamine Oxidases A and B" @default.
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