Role of the General Base Glu-268 in Nitroglycerin Bioactivation and Superoxide Formation by Aldehyde Dehydrogenase-2

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited ∼50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels. Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited ∼50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels. Aldehyde dehydrogenases (ALDH; EC 1.2.1.3) 2The abbreviations used are: ALDHmitochondrial aldehyde dehydrogenase (EC 1.2.1.3)cGMP3′,5′-cyclic guanosine monophosphateDEA/NO2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NONOate)DTTdithiothreitolGDNglyceryl dinitrateGTNglyceryl trinitrate (nitroglycerin)NADnicotinamide adenine dinucleotidesGCsoluble guanylate cyclaseSIN-13-morpholino sydnonimineTEAtriethanolamineSODsuperoxide dismutaseWTwild typeDTPAdiethylene triamine pentaacetic acid. catalyze the oxidation of aliphatic and aromatic aldehyde substrates to the corresponding carboxylic acids with NAD(P) serving as electron accepting co-factor (1Racker E. J. Biol. Chem. 1949; 177: 883-892Abstract Full Text PDF PubMed Google Scholar). The mitochondrial isoform (ALDH2), a homotetrameric protein with subunits of ∼54 kDa, appears to be essential for detoxification of ethanol-derived acetaldehyde, as indicated by significantly lowered alcohol tolerance of individuals expressing a low activity mutant of the protein (2Crabb D.W. Edenberg H.J. Bosron W.F. Li T.K. J. Clin. Invest. 1989; 83: 314-316Crossref PubMed Scopus (490) Google Scholar, 3Larson H.N. Zhou J. Chen Z. Stamler J.S. Weiner H. Hurley T.D. J. Biol. Chem. 2007; 282: 12940-12950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Aldehyde oxidation by ALDH2 is thought to involve nucleophilic reaction of the substrate with a critical cysteine residue in the active site (Cys-302 in the human protein), resulting in formation of a thiohemiacetal intermediate, followed by hydride transfer to NAD, yielding a thioester intermediate that is hydrolyzed to the carboxylic acid product in a reaction that involves activation of H2O by an adjacent glutamate residue (Glu-268). In addition to aldehyde oxidation, ALDH2 catalyzes ester hydrolysis (4Mann C.J. Weiner H. Prot. Sci. 1999; 8: 1922-1929Crossref PubMed Scopus (58) Google Scholar). The esterase activity is stimulated by NAD, but the co-factor is not essential for the reaction, which is initiated by nucleophilic attack of the substrate by Cys-302, resulting in formation of a thioester and release of the corresponding alcohol by hydrolysis of the intermediate through activation of water by Glu-268 (4Mann C.J. Weiner H. Prot. Sci. 1999; 8: 1922-1929Crossref PubMed Scopus (58) Google Scholar). mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) 3′,5′-cyclic guanosine monophosphate 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NONOate) dithiothreitol glyceryl dinitrate glyceryl trinitrate (nitroglycerin) nicotinamide adenine dinucleotide soluble guanylate cyclase 3-morpholino sydnonimine triethanolamine superoxide dismutase wild type diethylene triamine pentaacetic acid. The beneficial therapeutic effects of the antianginal drug GTN are thought to involve bioactivation of the organic nitrate in vascular smooth muscle to yield NO or a related species that activates sGC, resulting in cGMP-mediated vasorelaxation (5Fung H.L. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 67-85Crossref PubMed Scopus (119) Google Scholar). In a seminal paper published in 2002, Stamler and co-workers (6Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8306-8311Crossref PubMed Scopus (503) Google Scholar) discovered that ALDH2 essentially contributes to vascular GTN bioactivation, and this has been confirmed in numerous later studies (for review see Ref. 7Mayer B. Beretta M. Br. J. Pharmacol. 2008; 155: 170-184Crossref PubMed Scopus (95) Google Scholar). Stamler and co-workers (6Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8306-8311Crossref PubMed Scopus (503) Google Scholar) proposed that GTN denitration involves the established esterase activity of ALDH2, i.e. nucleophilic attack of a nitro group of GTN by Cys-302, resulting in formation of a thionitrate intermediate and release of the corresponding alcohol, preferentially 1,2-glyceryl dinitrate (1,2-GDN). The thionitrate intermediate would then release nitrite either through nucleophilic attack of one of the adjacent cysteine residues (Cys-301 or Cys-303), resulting in formation of a disulfide in the active site, or through Glu-268-aided hydrolysis yielding a sulfenic acid derivative of Cys-302, which could undergo S-thiolation (8Biswas S. Chida A.S. Rahman I. Biochem. Pharmacol. 2006; 71: 551-564Crossref PubMed Scopus (474) Google Scholar) to form a cysteinyl disulfide with one of the adjacent cysteine residues. This mechanism would be compatible both with the effect of NAD, which is not essential but increases reaction rates, and with GTN-triggered enzyme inactivation that is partially prevented by reduced thiols with two SH groups like DTT or dihydrolipoic acid. According to a brief statement in a paper on the structure of the East Asian (E487K) variant, mutation of Cys-302 and Glu-268 resulted in an almost complete loss of GTN reductase activity of ALDH2 (3Larson H.N. Zhou J. Chen Z. Stamler J.S. Weiner H. Hurley T.D. J. Biol. Chem. 2007; 282: 12940-12950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), but so far the proposed role of these residues in GTN metabolism has not been thoroughly studied, and the mechanism underlying bioactivation of the nitrate is still unknown. Bovine lung sGC was purified as previously described (9Russwurm M. Koesling D. Methods Enzymol. 2005; 396: 492-501Crossref PubMed Scopus (32) Google Scholar). Human ALDH2 was expressed in Escherichia coli BL21(DE3) and purified as described (10Zheng C.F. Wang T.T. Weiner H. Alcohol Clin. Exp. Res. 1993; 17: 828-831Crossref PubMed Scopus (67) Google Scholar, 11Beretta M. Gruber K. Kollau A. Russwurm M. Koesling D. Goessler W. Keung W.M. Schmidt K. Mayer B. J. Biol. Chem. 2008; 283: 17873-17880Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Sephacryl S-300 HR and [α-32P]GTP (400 Ci/mmol) were obtained from GE Healthcare. [2-14C]GTN (50–60 mCi/mmol) was from American Radiolabeled Compounds, purchased through Humos Diagnostica GmbH (Maria Enzersdorf, Austria). Nitropohl® ampoules (G. Pohl-Boskamp GmbH. & Co, Hohenlockstedt, Germany) containing 4.4 mm GTN in 250 mm glucose were obtained from a local pharmacy; dilutions were made in 50 mm TEA/HCl buffer. DEA/NO and SIN-1 were from Alexis Corporation (Lausen, Switzerland). All other chemicals, including hydralazine and CuZnSOD, were from Sigma-Aldrich. C302S and E268Q mutations were inserted using the QuikChange® II site-directed mutagenesis kit (Stratagene). The mutagenic primers 5′-GTTCTTCAACCAGGGCCAGTGCAGCTGTGCCGGATCCCGGACCTTCGTG-3′ (sense strand) and 5′-GAAGGTCCGGGATCCGGCACAGCTGCACTGGCCCTGGTTGAAGAAC-3′ (antisense strand) were used to introduce C302S mutation (bold type) and to add the BamHI restriction site (underlined) for a rapid screening of mutants but the acid The mutagenic primers and were used to introduce E268Q mutation and to add the restriction site as a mutation (underlined) for screening of and E268Q-ALDH2 were expressed in E. coli BL21(DE3) and purified by and as described M. Gruber K. Kollau A. Russwurm M. Koesling D. Goessler W. Keung W.M. Schmidt K. Mayer B. J. Biol. Chem. 2008; 283: 17873-17880Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). activity of ALDH2 was measured as formation of from NAD by the increase in in the presence of mm and mm in 50 mm or Keung W.M. PubMed Scopus Google Scholar). activity was measured by formation of from mm in 50 mm containing mm in the and presence of mm GTN-triggered inactivation of ALDH2 was in the and presence of as described M. A. Schmidt K. Mayer B. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar). the dehydrogenase activity of ALDH2 was measured by the formation of The reaction of mm and mm NAD in 50 mm in the or presence of the were by the addition of the of the 50 GTN was to the enzyme. complete mm DTT was to the enzyme. The increases the addition of ALDH2 and DTT were used to and activities, The inactivation GTN addition was to a of a and a with the yielding the inactivation and the yielding the activity of the enzyme. The rates of GTN metabolism yielding and were to a described A. A. Russwurm M. Koesling D. Keung W.M. Schmidt K. Mayer B. Biochem. J. 2005; PubMed Scopus Google Scholar). indicated WT and ALDH2 were with GTN for in a of of 50 mm containing mm and mm DTT in the and presence of mm GTN were by the addition of were with of by and by were in the of protein and The results are the in were not significantly by the two mutations described the rates of formation are not lung sGC was for or as indicated in a of with the indicated of and in the presence of WT- or E268Q-ALDH2 as indicated in the and 50 mm TEA/HCl mm [α-32P]GTP mm mm and mm NAD, and were as indicated in the and To for NO by sGC was with WT- or E268Q-ALDH2 in the presence of GTN and were as in the The were by the addition of of and of followed by and of as previously described E. J. M. Methods of Scholar). The were in the of NO formation was measured with a with nitrite as previously described B. Schmidt K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was in 50 mm TEA/HCl containing indicated mm mm mm and purified WT- or E268Q-ALDH2 followed by the addition of GTN The was was in The in was with of ALDH2 NO formation to formation in was from the rates of product formation catalyzed by WT- and E268Q-ALDH2 in the and presence of The of the were to Ref. Z. of of GTN-derived NO to denitration catalyzed by WT- and WT NAD and E268Q NAD of WT NAD and E268Q NAD of WT NAD and E268Q NAD of WT NAD and E268Q NAD of WT NAD and E268Q NAD of in a Formation of was measured as hydroethidine The reaction 50 mm mm mm mm WT- or E268Q-ALDH2 to and indicated mm mm chloral and The were and was and of and The are the of the increase in in to and E268Q-ALDH2 were for oxidation of and hydrolysis of and denitration of GTN to in mutation of the reactive cysteine residue Cys-302 resulted in loss of dehydrogenase and esterase activities. GTN reductase activity of the C302S mutant of may be to reaction of GTN with one of the adjacent cysteine residues. of Glu-268 caused virtually complete loss of dehydrogenase but determination of the esterase activity in the and presence of NAD an effect of the E268Q Although NAD caused an increase of WT esterase the activity of the E268Q mutant was in the presence of the nicotinamide co-factor by The effects of NAD on WT- and E268Q-ALDH2 were also in of GTN denitration. Although both of the enzyme exhibited virtually rates of formation in the of NAD, the E268Q mutation resulted in an almost complete loss of GTN activity of in the presence of ester GTN denitration catalyzed by E268Q-ALDH2 was by the and GTN reductase activities of and to to in a In the presence of NAD, GTN was by WT-ALDH2 with an of rates of corresponding to a activity of were previously A. A. Russwurm M. Koesling D. Keung W.M. Schmidt K. Mayer B. Biochem. J. 2005; PubMed Scopus Google Scholar), the reaction rates measured low GTN were lower in the of NAD, the nicotinamide a effect in the presence of GTN In the of NAD the of GTN was to In to the WT E268Q-ALDH2 not catalyze formation in the presence of NAD GTN In the of the the mutant exhibited rates of GTN denitration as the WT enzyme the GTN was obtained the rates of GTN denitration were measured as a function of the protein of in formation in a ALDH2 to 50 the of the WT enzyme product formation rapid enzyme inactivation activity of WT-ALDH2 measured with of in the presence of NAD of was This is than the activity measured a on enzyme inactivation that is partially prevented by DTT M. A. Schmidt K. Mayer B. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar). The activity of the WT enzyme was markedly by NAD, the activity of the mutant was all of the protein of the mutant activity in the presence of NAD, resulting in pronounced of GTN denitration by the previously M. Gruber K. Kollau A. Russwurm M. Koesling D. Goessler W. Keung W.M. Schmidt K. Mayer B. J. Biol. Chem. 2008; 283: 17873-17880Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the presence of is essential for of GTN-derived NO by as in the E268Q mutant of NO in the of of reduced superoxide in the of GTN The of NO formation by E268Q-ALDH2 as compared with WT in the presence of that the E268Q led to significantly NO rates of enzyme To see the between and NO formation was by the E268Q NAD, we measured the rates of NO formation catalyzed by of WT- and in the presence of with and NAD and compared the with the rates of formation in in the rates of NO formation catalyzed by WT-ALDH2 were between and of the denitration rates, and product was not significantly by the E268Q mutant an Although NO formation was to WT in the of NAD, the of GTN reductase activity was not by a in the rates of NO formation in the presence of the resulting in a increase of NO formation to of the denitration rates the of protein in sGC was by GTN in the presence of of but the effect a of of sGC activity of ALDH2 In the presence of mm the rates of formation were but a of sGC activation was with of protein To sGC activation is limited by co-generation of which with NO to yield we measured GTN-triggered in the presence of markedly potentiated the effect of ALDH2, resulting in sGC activation with of and DTT the rates of formation by that the rates of formation measured with E268Q-ALDH2 in the of DTT were than with WT all protein In the presence of the effect of the mutant was virtually to that of a pronounced between the two ALDH2 sGC activation was measured in the presence of DTT of a of sGC activity with E268Q-ALDH2 and sGC activation was with of the protein in the of in this between the two ALDH2 was also as formation of GTN-derived NO by the E268Q mutant but not by the WT enzyme in the of The in between and the SIN-1 in of sGC activation and NO release SIN-1 NO than the The underlying this was not The also that the of the NO is low to the of NO that are for sGC the effect of GTN on sGC activation with WT- or E268Q-ALDH2 in the presence of mm GTN sGC in the presence of WT-ALDH2 with an of The E268Q mutant was to WT in the presence of to GTN but of the sGC activation in the presence of GTN in the of The of GTN was to denitration catalyzed by the E268Q mutant in the of led to pronounced of the GTN in the presence of both WT- and The effect of NAD on GTN-triggered sGC activation was measured in in the presence of to with the and NO in NAD effect on formation in the presence of either WT-ALDH2 or GTN and sGC to GTN the rates of formation measured in the presence of WT-ALDH2 were by NAD, the nucleotide significantly inhibited sGC activation in the presence of the E268Q mutant. The pronounced effect of on GTN-triggered sGC activation that ALDH2-catalyzed GTN metabolism is with superoxide we for of the reaction with sGC activation by NO. in GTN WT- or E268Q-ALDH2 effect but the presence of WT enzyme and GTN resulted in reduction of sGC activity that was by Although NO was also prevented by the hydralazine A. M. M. K. S. M. A. Biochem. Res. 2005; PubMed Scopus Google Scholar), the and mm effect not In to reaction of GTN with the E268Q mutant not NO that GTN catalyzed by WT-ALDH2 resulted in oxidation of formation of reactive species oxidation was significantly inhibited by chloral and and reduced by ∼50% GTN was with the E268Q mutant of the WT enzyme. hydroethidine oxidation was not significantly inhibited by suggesting the of reactive species other than that formation of superoxide in the of GTN denitration could contribute to inactivation of as in of superoxide by effect on the rates of and ALDH2 inactivation measured as oxidation in the presence of GTN and the addition of This and of ALDH2-catalyzed bioactivation of is essential for aldehyde oxidation and ester hydrolysis but not for GTN GTN bioconversion is inhibited by NAD, and the reaction results in superoxide formation that NO bioavailability and is significantly reduced mutation of In Stamler and co-workers (6Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 8306-8311Crossref PubMed Scopus (503) Google Scholar) proposed a reaction of GTN with the nucleophilic Cys-302 residue of ALDH2 as the of GTN Our results with this GTN reductase activity of the mutant of of other nucleophilic or to the The loss of aldehyde dehydrogenase and esterase activities mutation of Glu-268 with a that this residue is essentially involved in hydride activation of Cys-302, hydrolysis of thioester Hurley T.D. PubMed Scopus Google Scholar), of GTN denitration measured in the presence of NAD (3Larson H.N. Zhou J. Chen Z. Stamler J.S. Weiner H. Hurley T.D. J. Biol. Chem. 2007; 282: 12940-12950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Although the presence of NAD as hydride is for the dehydrogenase reaction, ester hydrolysis also in the of NAD, that the nucleotide is not an essential co-factor but reaction rates through a of the active site NAD not but inhibited the esterase activity of E268Q-ALDH2 by The effects of NAD were also in GTN denitration reaction rates of WT- and E268Q-ALDH2 in the of NAD, the nicotinamide the activity of WT but GTN denitration by the E268Q mutant. The effect of NAD on WT-ALDH2 was of an increase in GTN resulting in by the nucleotide with GTN as previously A. A. Russwurm M. Koesling D. Keung W.M. Schmidt K. Mayer B. Biochem. J. 2005; PubMed Scopus Google Scholar). the the effect of NAD was not by but protein the E268Q exhibited activity in the presence of In the form of WT-ALDH2 two of been T.D. S. H. Chem. Biol. PubMed Scopus Google in the the Cys-302, is in the The is to be by and a water between the and the thioester intermediate Hurley T.D. PubMed Scopus Google Scholar). the active in the the Glu-268 in the and in the The NAD is the nicotinamide in the hydrolysis all Glu-268 are in the active and Hurley T.D. PubMed Scopus Google Scholar). This in the in of the active could the increase in esterase effects on the activity caused by of the esterase substrate or the intermediate with the co-factor be also been for the nicotinamide in of ALDH2 with In one transfer the is to Cys-302, in the the has the to the active has been that NAD be in both the In the the nicotinamide is between Glu-268 and Cys-302, which is compatible with hydride transfer the but with hydrolysis of an enzyme intermediate The to the other NAD Hurley T.D. PubMed Scopus Google Scholar). for the of the activity of the E268Q of ALDH2 NAD a in the between the two co-factor the hydride transfer is by the enzyme the hydrolysis this would to the activity In Glu-268 a to the nicotinamide in the hydride transfer a this co-factor ester could be by for a compared with a glutamate two with the of the Formation of with GTN bioactivation as NO release or sGC activation in the presence of suggesting that and NO are from a reaction intermediate, a thionitrate Cys-302 to one of the adjacent cysteine residues. The mechanism of NO formation is but that the thionitrate intermediate is through two that both yield but either nitrite or NO as The reduction yielding NO appears to for of GTN catalyzed by in the reaction catalyzed by E268Q-ALDH2 in the presence of NAD, NO formation to to of suggesting that the two are not Although this pronounced increase in NO formation of low rates of GTN was between the rates of GTN and the of GTN to NO. we that the between the and reduction of GTN denitration appears to be by the active site structure Glu-268 of human The in the presence of of GTN that ALDH2-catalyzed reduction of GTN to yield NO vascular GTN in this may not be the complete to GTN-derived NO in blood M. A. H. E. K. B. A. Res. PubMed Google Scholar, A. S. Res. 2005; PubMed Scopus Google Scholar, S. J. 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