Structural Comparison of Human Monoamine Oxidases A and B

MASS SPECTROMETRY MONITORING OF CYSTEINE REACTIVITIES*

Frantisek Hubálek {ddagger}, Jan Pohl § and Dale E. Edmondson {ddagger} 

From the {ddagger}Department of Biochemistry and the §Microchemical and Proteomics Facility, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, April 9, 2003 , and in revised form, May 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({tau}1/2 = ~3 min) than MAO B ({tau}1/2 = ~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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Monoamine oxidases (MAO)1 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 (1). 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 (1, 2). 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% (3). Activity of either enzyme is sensitive to thiol-modifying reagents (4, 5), and cysteine residues have been implicated in the catalytic mechanism of MAO based on the results of site-directed mutagenesis (6, 7), thiol titration (8), and inhibitor binding (9) 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) (3).2 In each enzyme, one conserved cysteine residue (Cys406 in MAO A and Cys397 in MAO B) is linked in a thioether bond to the 8{alpha}-methylene of FAD (10, 11), therefore leaving 8 cysteine residues either as free thiols or in disulfide bonds. Gomes et al. (8) 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 (9) by demonstrating this residue to be modified on inhibition of bovine liver MAO B with N-cyclopropyl-{alpha}-methylbenzylamine. Mutagenesis experiments by Wu et al. (6) 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 (6). Cesura et al. (7) found that MAO B activity is decreased significantly when Cys365 is mutated to Ala. The recently solved crystal structure of MAO B (12) reveals that Cys365 and Cys156, previously suggested to be involved in catalytic mechanism (6, 7, 9), are located on the surface of the molecule.



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SCHEME 1.
All sequences were obtained from Swiss-Prot data base: human MAO B (P27338 [GenBank] ), rat MAO B (P19643 [GenBank] ), guinea pig MAO B (P58028 [GenBank] ), bovine MAO A (P21398 [GenBank] ), dog MAO A (P58027 [GenBank] ), guinea pig MAO A (P58028 [GenBank] ), rat MAO A (P21396 [GenBank] ), mouse MAO A (Q64133 [GenBank] ), and human MAO A (P21397 [GenBank] ). The top numbers represent the cysteine positions in human MAO B, and the bottom numbers represent the cysteine positions in human MAO A.

 

A recent study (13) 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 (14, 15).

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 (14) and MAO B (15) 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 (1618). The level of resolution achieved by electrospray ionization quadrupole mass spectrometry allows for monitoring of the distribution of reacted thiol groups in the enzymes (19). 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All buffers and reagents were purchased in the highest grade available from Sigma unless specified otherwise. {beta}-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 (14, 15); 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 x 20 cm) in 50 mM potassium phosphate buffer, pH 7.5, containing 0.8% (w/v) {beta}-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 (14, 15). 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 (14) or the benzylamine assay for MAO B (15), 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 x 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 (20). 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 x 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 x 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 (21) 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Disulfide Bond Analysis—MAO A and MAO B each contain 9 cysteine residues (Scheme 1) (3). One cysteine residue (A407/B397) is covalently attached to FAD (10, 11), 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.



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FIG. 1.
Disulfide bond analysis of human recombinant MAO A and B. ESI-MS spectra of human recombinant MAO A and MAO B incubated with N-ethylmaleimide in the presence of guanidine HCl under reducing (B for MAO A, observed mass= 61,510 ± 8 Da, calculated mass = 61,511 Da, and E for MAO B, observed mass = 60,473 ± 14 Da, calculated mass = 60,458 Da) or nonreducing conditions (C for MAO A, observed mass = 61,520 ± 14 Da, calculated mass = 61,511 Da, and F for MAO B, observed mass = 60,473 ± 12 Da, calculated mass = 60,458 Da) are shown. In both cases, MAO masses increased by 1000 Da relative to the mass of unreacted enzymes (A for MAO A, observed mass = 60,512 ± 8 Da, calculated mass = 60,510 Da, and D for MAO B, observed mass = 59,475 ± 14 Da, calculated mass = 59,458 Da), corresponding to the addition of 8 molecules of NEM.

 

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 {tau}1/2 of ~2 min, whereas MAO B is inactivated with a {tau}1/2 of ~8 h under the same conditions. At 0 °C, MAO A is inactivated with a {tau}1/2 of ~3 h, and no inactivation of MAO B is observed ({tau}1/2 > 100 h). MAO B is inactivated at a faster rate ({tau}1/2 = ~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 (13).


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TABLE I
Half-times for MAO A and MAO B inactivation with NEM and Biotinyl NEM

 

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).



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FIG. 2.
Influence of clorgyline and pargyline inactivation on NEM modification of MAO A and B. Deconvoluted ESI-MS spectra of both MAO A and MAO B after incubation with an 80-fold excess of NEM are shown. Clorgyline inactivation protected ~3 cysteine residues in MAO A from NEM modification at both temperatures. Pargyline inactivation had little effect on the reactivities of MAO B cysteine residues at 0 °C but prevented the high stoichiometry modifications at 25 °C. The peaks are labeled with the number of NEM groups bound to the enzyme, and the incubation times and temperatures are indicated.

 

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 (22). 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 {tau}1/2 = ~5 min at 25 °C and {tau}1/2 = ~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 ({tau}1/2 > 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.



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SCHEME 2.
EZ-link Biotinyl-NEM (molecular mass = 525.2 Da).

 


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FIG. 3.
Influence 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.

 

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 (23), 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.3



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FIG. 4.
SELDI-MS analysis of biotinylated tryptic peptides from Biotinyl-NEM modified clorgyline-inactivated MAO A and pargyline-inactivated MAO B recovered during streptavidin affinity chromatography. Peptide 240–267 (LNHPVTHVDQSSDNIIIETLNHEHYECK, maleamic acid form, observed mass = 3827.9 Da, calculated mass = 3829.8 Da; maleimide form, observed mass = 3812.1 Da, calculated mass = 3811.8 Da), containing Cys266, was identified as the only Biotinyl-NEM-modified peptide in clorgyline-inactivated MAO A after 24 h of incubation at 0 °C (top panel). Peptide 5–21 (CDVVVVGGGISGMAAAK, maleimide form, observed mass = 2059.9 Da, calculated mass = 2059.0 Da; maleamic acid form, observed mass = 2077.7 Da, calculated mass = 2077.0 Da), containing Cys5, was identified as the only Biotinyl-NEM-modified peptide in MAO B (and pargyline-inactivated MAO B) after 24 h of incubation at 0 °C (bottom panel). Internal standard (ALSEGC(Biotinyl-NEM)TPYDIN, maleamic acid form, observed mass = 1825.5 Da, calculated mass = 1825.6 Da) was added to the MAO B sample prior to streptavidin chromatography. None of the remaining minor species correspond to a biotinylated MAO tryptic peptide.

 

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 (12) 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 (24). 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.



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FIG. 5.
Location of Cys5 in the crystal structure of MAO B and predicted location of Cys266 in a model of MAO A structure. The MAO A structure (right panel) was generated by replacing Cys5 and Ala257 in the crystal structure of MAO B with Phe14 and Cys266, respectively (12). A ribbon diagrams of parts of MAO B (left panel) and MAO A(right panel) structures opposite to the membrane-binding regions are shown. Cys5 and Ala257 in MAO B and Cys266 and Phe14 in MAO A are space-filled. The asterisks mark the sulfur atoms of the cysteine residues. This figure was created using a PyMol Molecular Graphics System (www.pymol.org).

 

Analysis of MAO A and MAO B in Intact Mitochondria—To determine whether the conclusions from the above experiments performed with detergent-solubilized enzymes (in vitro) hold true for the membrane-bound forms, we performed similar modification experiments using intact mitochondria from P. pastoris overexpressing either enzyme. In this expression system, >95% of either enzyme produced is bound to the yeast mitochondrial membrane (data not shown). SDS-PAGE analysis of the intact mitochondria shows that either overexpressed enzyme accounts for >80% of the total mitochondrial protein (Fig. 6). Cysteine residues of either enzyme react with Biotinyl-NEM when intact mitochondria are incubated with the reagent as demonstrated by Western blots of mitochondrial proteins developed with streptavidin conjugated to alkaline phosphatase (Fig. 6). In addition to MAO (that represented the most intense band), as expected, several other proteins were also biotinylated, including a ~90-kDa protein. Given the relative intensities of the protein and biotin staining, the stoichiometry of Biotinyl-NEM modification of MAO (1:1) is low relative to the 90-kDa protein. This is probably due to a larger number of cysteine residues in the 90-kDa protein that are accessible to Biotinyl-NEM modification. No attempts were made to identify the 90-kDa protein.



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FIG. 6.
SDS-PAGE and streptavidin Western blot analysis of MAO A and MAO B in intact mitochondria. Mitochondrial proteins isolated from P. pastoris overexpressing MAO A (lane 1) and MAO B (lane 2) were separated by SDS-PAGE using a 7.5% resolving gel and stained with Coomassie Brilliant Blue (6 µg of protein/lane). Purified MAO B was used as a positive control (2 µg, lane 3). Streptavidin Western blot analysis of mitochondria incubated with Biotinyl-NEM isolated from P. pastoris overexpressing MAO A (lane 6) and MAO B (lane 5) are shown. Purified biotinylated MAO A (lane 7) and MAO B (lane 4) are used as positive controls. Aliquots of 0.01 µg of protein were used for each lane.

 

If the MAO structure in the mitochondrial membrane is the same as the structure of purified enzyme in detergent solution, then the same cysteine residues should react with Biotinyl-NEM in either case. Indeed, a biotinylated MAO A peptide 240–267 containing Cys266 was identified using SELDI MS analysis of a MAO A in-gel tryptic digest (Fig. 7). However, no biotinylated peptide could be identified by SELDI analysis of a MAO B in-gel tryptic digest (Fig. 7). The expected peptide 5–21 is likely to be extracted from the gel in very low yields because of its hydrophobicity (CDVVVVGGGISGMAAAK). However, evidence of this biotinylated MAO B peptide containing Cys5 is obtained by using selective ion monitoring LC-ESI-MS of the MAO B in-gel tryptic digest (Fig. 7). Peptides containing Cys266 and Cys5 were the only biotinylated peptides identified in mitochondrial membrane-bound clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively, similar to the results obtained for detergent-solubilized purified enzymes. These results provide experimental evidence suggesting that MAO A and MAO B structures in outer mitochondrial membrane are similar to their structures in detergent solutions.



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FIG. 7.
Analysis of biotinylated tryptic peptides recovered from streptavidin affinity chromatography of mitochondrial membrane-bound clorgyline-inactivated MAO A and pargyline-inactivated MAO B modified with Biotinyl-NEM. Peptide 240–267 containing Cys266 (labeled with arrows, observed mass = 3810.2 Da, maleimide form; observed mass = 3829.6 Da, maleamic acid form) was identified as the only Biotinyl-NEM-modified peptide in clorgyline-inactivated MAO A after 15 h of incubation at 4 °C (A). Major species in the spectrum are interpreted as unmodified MAO A peptides: peptide 281–297, observed mass = 2115.1 Da, calculated mass = 2116.4 Da and peptide 110–129, observed mass = 2409.6 Da, calculated mass = 2407.7 Da. No biotinylated peptide was observed in similar analysis of pargyline-inactivated MAO B (B). The expected position of biotinylated MAO B peptide 5–21 is indicated with an arrow. Major species in the spectrum are interpreted as unmodified MAO B peptides: peptide 21–35, observed mass = 1637.7 Da, calculated mass = 1635.9 Da and peptide 100–119, observed mass = 2474.4 Da, calculated mass = 2471.8 Da. Both forms (maleamic acid form, 69.4 min, and maleimide form, 72.8 min) of Biotinyl-NEM MAO B peptide 5–21 were identified during selective ion monitoring (m/z = 1031.0 and 1039.9 for the doubly charged ions, and m/z = 687.6 and 693.6 for the triple charged ions of the two forms respectively) LC-MS experiment (C). Deconvoluted ESI-MS spectra for the maleamic acid (D) and the maleimide (E) forms of the biotinylated MAO B peptide 5–21 are shown. Sequence information and calculated masses of the peptides are reported in legend of Fig. 4.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of the thiol groups in the structure and function of MAO A and MAO B has been the subject of numerous investigations since the early work of Barron and Singer (5). Most of the studies have concluded that thiol group(s) are at the catalytic site and/or involved in the catalytic mechanism (69). This conclusion followed the well established paradigm in enzymology that protection of group reactivity by a substrate or a competitive inhibitor provides strong evidence for that group to be located in the catalytic site. The recent determination of the three-dimensional structure of MAO B (12) provides direct evidence that, in fact, there are no identifiable thiol groups in the catalytic site of MAO B and lead to the conclusion that other factors contribute to the strikingly different reactivities of the thiol groups in these enzymes.

To investigate thiol group reactivities in a ~60-kDa protein at the sequence level, an approach was required that would yield high resolution and enable direct identification of modified cysteine residues. A second consideration was the very hydrophobic nature of the resulting tryptic, Cys-containing peptides. The first requirement was satisfied by modifying the thiol groups with a reagent that exhibits a high level of specificity and selectivity; N-ethylmaleimide was chosen because this reagent exhibits a good selectivity for thiol groups. Although modification of histidine, lysine, and tyrosine residues have been observed at high reagent concentrations and long reaction times (22), the selectivity of NEM for cysteine residues is still superior as compared with the alternatives, which include haloacyl- or acryloyl-based reagents. ESI quadrupole MS was applied because it provides a high resolution allowing us to monitor additions of 125 Da to a ~60-kDa protein (19). Using MAO A and MAO B, we demonstrated that this approach is feasible and provides a better description of the relative levels of different populations of reaction products as compared with more traditional approaches, such as radiolabeling or spectrophotometric monitoring of the reaction.

The findings of no detectable disulfide bonds in MAO A or MAO B are in accord with x-ray data on MAO B (12), which showed the shortest distance between two thiol groups to be ~7.5 Å, i.e. much greater than a typical S–S distance (~2.2 Å). No disulfide bonds were identified in MAO A despite the fact that two cysteine residues, Cys321 and Cys323, are present in close proximity in the primary sequence. The findings of no disulfide bonds in either enzyme do not support the conclusions of Sablin and Ramsay (13), which proposed the presence of redox active disulfide bonds in both enzymes on the basis of reducing equivalents required for flavin reduction upon dithionite titrations. Stoichiometric amounts of substrates were previously found sufficient for full reduction of either enzyme (14, 15). The lack of disulfide bonds in either enzyme is also consistent with the results of Nandigama and Edmondson (25), which demonstrated that apoMAO A can be inserted into the outer mitochondrial membrane in the absence of flavin in the rib5 strain of Saccharomyces cerevisiae riboflavin auxotroph, and the activity of this enzyme is restored upon the addition of exogenous FAD. It had previously been demonstrated that yeast thiol/disulfide isomerase is a flavoprotein (26) and would not be active in the absence of exogenous flavin and therefore would not be able to catalyze disulfide bond formation in newly synthesized proteins.

The reaction of MAO A with NEM proceeds rapidly to form a species with 7 or 8 modified thiol groups. These data suggest that after the initial alkylation of a few thiol groups on the enzyme with NEM, the enzyme structure unfolds to one in which all of the thiol groups are now reactive with the thiol reagent (Fig. 3). This behavior is also observed with the larger Biotinyl-NEM reagent where the major species observed with MAO A is either an unmodified enzyme or a species with 5 or 7 Biotinyl-NEM molecules added. The reactivity profile of MAO B with this reagent suggests a more stable structure where extensive reaction with NEM occurs only on prolonged incubation. These data are consistent with the relative stabilities of the two enzymes observed by investigators who have worked with both purified enzymes.

A covalent modification of the active site flavin by acetylenic amines (16) leads to reduced stoichiometry of NEM or Biotinyl-NEM modification of MAO A and MAO B (Figs. 3 and 4). These findings demonstrate that binding of an inhibitor to the catalytic site of either enzyme results in a pronounced stabilization of their respective structures. Thus, the protective effect of substrate/inhibitor binding to thiol reagents observed by others can be attributed to this stabilizing influence of the three-dimensional fold of the protein(s) structure rather than steric hindrance of the active site thiol group by the bound substrate/inhibitor. There are no detectable changes in the {alpha}-helical content of either MAO A or MAO B upon acetylenic inhibitor binding as judged by UV circular dichroism studies (27). The {alpha}-helical contents of both enzymes are similar (estimated at ~45%) and in excellent agreement with the {alpha}-helical content calculated based on MAO B x-ray structure (44%) (12, 27). The similar estimated helical contents of MAO A and MAO B predict that both enzymes have similar three-dimensional structures. This conclusion is consistent with their thiol reactivity profiles (Fig. 5).

The observation that Biotinyl-NEM reacts with the same cysteinyl residues in the membrane-bound and detergent-solubilized forms of MAO A and MAO B supports the view that the C-terminal helix of MAO B serves as the major anchor of the protein in the outer mitochondrial membrane, and the N-terminal part of the enzyme is in a solvent accessible environment. Although our experiments were performed with overexpressed human enzymes in P. pastoris, we suggest that our findings are relevant to the natural environment of the enzyme because the extraction of the enzymes from yeast mitochondria requires conditions similar to those used when extracting the enzymes from either beef liver or human placental mitochondria (14, 15). The substantial permeability of the outer membrane precludes any conclusions regarding the orientation of the enzymes with respect to facing the cytoplasmic or the inner membrane compartments. It is worthwhile to note that although both Cys266 and Cys5 are conserved in all known MAO A and MAO B sequences, respectively (Scheme 1), Ser mutation of either Cys residue has no effect on enzyme activity (6).


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM-29433. The mass spectrometry equipment of the Emory University Microchemical Facility used throughout this work was purchased by National Institutes of Health shared Instrumentation Grants NCRR-02878, NCRR-12878, and NCRR-13948. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, Rollins Research Bldg., 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5972; Fax: 404-727-3452; E-mail: dedmond{at}bimcore.emory.edu.

1 The 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. Back

2 Numbering for both enzymes refers to the gene-predicted amino acid sequences. On the protein level, the initial Met residue of MAO B is removed. Back

3 This 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). Back


    ACKNOWLEDGMENTS
 
We are grateful to Min Li and Dr. P. Newton-Vinson for providing purified preparations of recombinant MAO A and MAO B.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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