Methionine Adenosyltransferase S-Nitrosylation Is Regulated by the Basic and Acidic Amino Acids Surrounding the Target Thiol

Abstract

S-Adenosylmethionine serves as the methyl donor for many biological methylation reactions and provides the propylamine group for the synthesis of polyamines.S-Adenosylmethionine is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase. The cellular factors regulating S-adenosylmethionine synthesis have not been well defined. Here we show that in rat hepatocytesS-nitrosoglutathione monoethyl ester, a cell-permeable nitric oxide donor, markedly reduces cellularS-adenosylmethionine content via inactivation of methionine adenosyltransferase by S-nitrosylation. Removal of the nitric oxide donor from the incubation medium leads to the denitrosylation and reactivation of methionine adenosyltransferase and to the rapid recovery of cellular S-adenosylmethionine levels. Nitric oxide inactivates methionine adenosyltransferase viaS-nitrosylation of cysteine 121. Replacement of the acidic (aspartate 355) or basic (arginine 357 and arginine 363) amino acids located in the vicinity of cysteine 121 by serine leads to a marked reduction in the ability of nitric oxide to S-nitrosylate and inactivate hepatic methionine adenosyltransferase. These results indicate that protein S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target cysteine. S-Adenosylmethionine serves as the methyl donor for many biological methylation reactions and provides the propylamine group for the synthesis of polyamines.S-Adenosylmethionine is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase. The cellular factors regulating S-adenosylmethionine synthesis have not been well defined. Here we show that in rat hepatocytesS-nitrosoglutathione monoethyl ester, a cell-permeable nitric oxide donor, markedly reduces cellularS-adenosylmethionine content via inactivation of methionine adenosyltransferase by S-nitrosylation. Removal of the nitric oxide donor from the incubation medium leads to the denitrosylation and reactivation of methionine adenosyltransferase and to the rapid recovery of cellular S-adenosylmethionine levels. Nitric oxide inactivates methionine adenosyltransferase viaS-nitrosylation of cysteine 121. Replacement of the acidic (aspartate 355) or basic (arginine 357 and arginine 363) amino acids located in the vicinity of cysteine 121 by serine leads to a marked reduction in the ability of nitric oxide to S-nitrosylate and inactivate hepatic methionine adenosyltransferase. These results indicate that protein S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target cysteine. In the liver S-adenosylmethionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosylmethionine; NO, nitric oxide; MAT, methionine adenosyltransferase; SNO, nitrosothiol; EGSH, glutathione monoethyl ester; GSNO, S-nitrosoglutathione; EGSNO, S-nitrosoglutathione monoethyl ester; WT, wild type; NOx, nitrite and nitrate 1The abbreviations used are: AdoMet, S-adenosylmethionine; NO, nitric oxide; MAT, methionine adenosyltransferase; SNO, nitrosothiol; EGSH, glutathione monoethyl ester; GSNO, S-nitrosoglutathione; EGSNO, S-nitrosoglutathione monoethyl ester; WT, wild type; NOx, nitrite and nitrate serves as the methyl donor for many biological methylation reactions (such as DNA, proteins, phospholipids, and adrenergic, dopaminergic, and serotoninergic molecules) and provides the propylamine group for the synthesis of polyamines (1Cantoni G.L. Annu. Rev. Biochem. 1975; 44: 435-451Crossref PubMed Scopus (405) Google Scholar, 2Kotb M. Geller A.M. Pharmacol. Ther. 1993; 59: 125-143Crossref PubMed Scopus (114) Google Scholar, 3Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 1-16Crossref PubMed Scopus (400) Google Scholar). AdoMet is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase (MAT). There are two MAT genes; one is expressed only in the liver, and the other is expressed in extrahepatic tissues and fetal liver (2Kotb M. Geller A.M. Pharmacol. Ther. 1993; 59: 125-143Crossref PubMed Scopus (114) Google Scholar, 3Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 1-16Crossref PubMed Scopus (400) Google Scholar, 4Kotb M. Mudd S.H. Mato J.M. Geller A.M. Kredich N.M. Chou J.Y. Cantoni G.L. Trends Genet. 1997; 13: 51-52Abstract Full Text PDF PubMed Scopus (180) Google Scholar). Up to 85% of all methylation reactions and as much as 50% of methionine metabolism occur in the liver (5Mudd S.H. Poole J.R. Metabolism. 1975; 24: 721-735Abstract Full Text PDF PubMed Scopus (390) Google Scholar), which agrees with this tissue having the highest specific activity of MAT (6Finkelstein J.D. J. Nutr. Biochem. 1990; 1: 228-237Crossref PubMed Scopus (1076) Google Scholar). Moreover, in the liver the half-life of AdoMet is of only about 5 min (6Finkelstein J.D. J. Nutr. Biochem. 1990; 1: 228-237Crossref PubMed Scopus (1076) Google Scholar). Reduced levels of AdoMet and/or MAT activity, resulting in the abnormal metabolism of methionine, have been found in human cirrhosis and in a variety of experimental models including liver injury induced by ethanol, CCl4, and galactosamine (3Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 1-16Crossref PubMed Scopus (400) Google Scholar, 7Mato J.M. Alvarez L. Corrales F. Pajares M.A. Arias I.M. Boyer J.L. Fausto N. Schaachter D.A. The Liver Biology and Pathobiology. Raven Press, Ltd., New York1994: 461-470Google Scholar). The importance of this alteration in AdoMet synthesis in the pathogenesis of a variety of liver disorders is suggested by the finding that exogenous AdoMet administration protects from the hepatotoxic effect induced by a variety of agents, such as ethanol, CCl4, paracetamol, tumor necrosis factor, and galactosamine (3Mato J.M. Alvarez L. Ortiz P. Pajares M.A. Pharmacol. Ther. 1997; 73: 1-16Crossref PubMed Scopus (400) Google Scholar, 7Mato J.M. Alvarez L. Corrales F. Pajares M.A. Arias I.M. Boyer J.L. Fausto N. Schaachter D.A. The Liver Biology and Pathobiology. Raven Press, Ltd., New York1994: 461-470Google Scholar). The cellular factors regulating hepatic AdoMet levels are beginning to be defined. One such factor is nitric oxide (NO). In previous studies we demonstrated that conditions that induce NO synthesis, such as septic shock and hypoxia, induce the inactivation of hepatic MAT without affecting the expression of the liver-specific MAT gene (8Avila M.A. Mingorance J. Martı́nez-Chantar M.L. Casado M. Martı́n-Sanz P. Boscá L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar, 9Avila M.A. Carretero M.V. Rodrı́guez E.N. Mato J.M. Gastroenterology. 1998; 114: 364-371Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Further, we have reported previously that incubation of rat hepatocytes withS-nitrosoglutathione monoethyl ester (EGSNO), a cell-permeable NO donor, induces MAT inactivation (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). We have also shown that purified rat liver MAT is inactivated by incubation with NO donors (3-morpholinosydnonimine,S-nitroso-N-acetylpenicillamine, andS-nitrosoglutathione) (8Avila M.A. Mingorance J. Martı́nez-Chantar M.L. Casado M. Martı́n-Sanz P. Boscá L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar, 10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). In addition, we have recently demonstrated that liver MAT is S-nitrosylated both in vitro and in vivo (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), and further, we have identified cysteine 121 as the site of molecular interaction of NO and liver MAT (8Avila M.A. Mingorance J. Martı́nez-Chantar M.L. Casado M. Martı́n-Sanz P. Boscá L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar). Because these results indicate that NO (or related molecules) is a critical regulator of liver MAT activity, we were interested in whether AdoMet content in hepatocytes is regulated by NO. There are few studies trying to identify the active site features that control protein S-nitrosylation. It has been recently proposed (11Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar) that protein S-nitrosylation involves an acid-base-catalyzed nitrosothiol (SNO)/SH exchange reaction, where the target cysteine residue is localized next to basic and acidic amino acids. Here, we were interested in determining the structural factors that govern liver MAT S-nitrosylation and inactivation using liver recombinant enzyme and mutants of MAT where the acidic and basic amino acids in the vicinity of cysteine 121 were replaced by serine by site-directed mutagenesis. Recognition of the topology involved in protein S-nitrosylation is likely to prove useful in identifying new targets of protein S-nitrosylation. Hepatocytes were isolated from normally fed Wistar rats (250 g) as described previously (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). Isolated hepatocytes were incubated in the absence or presence of EGSNO at 37 °C. At the indicated periods of time, 2 ml of the cell suspension (2 × 106 cells/ml) were poured into precooled centrifuge tubes and washed twice with phosphate/saline buffer. Hepatocytes were then used for AdoMet or MAT activity determinations. Cell viability was determined before and after incubations by the trypan blue exclusion test. Only preparations with viability over 85% were used. AdoMet concentration was determined by high pressure liquid chromatography following the procedure described by Fell et al. (12Fell D. Benjamin L.E. Steele R.D. J. Chromatogr. 1985; 345: 150-156Crossref PubMed Scopus (51) Google Scholar) modified by Miller et al.(13Miller J.W. Nachau M.R. Smith J. Smith D. Selhub J. Biochem. J. 1994; 298: 415-419Crossref PubMed Scopus (221) Google Scholar). Samples of 2 × 106 hepatocytes washed twice in phosphate/saline buffer were homogenized in 200 μl of 0.4m perchloric acid. Homogenates were centrifuged at 10,000 × g and 4 °C for 15 min. 100 μl of the supernatant were analyzed on a Bio-Sil® ODS-5S column equilibrated in 0.01 m ammonium formate, 4 mmheptanesulfonic acid, pH 4.0. Elution was carried out with a 31-min linear gradient of acetonitrile (0–25%) in the same buffer. Chromatograms were analyzed using the Beckman System Gold software. Mutants are identified by a number that indicates the residue in the sequence of the enzyme that has been replaced. MAT mutants C121S, D355S, D355E, R357S, R363S, R363K, G359S, and R357S/R363S were obtained by inverse polymerase chain reaction according to the procedure of Serrano et al. (14Serrano L. Horovitz A. Avron B. Bycroft M. Fersht A.R. Biochemistry. 1990; 29: 9343-9352Crossref PubMed Scopus (357) Google Scholar) using the plasmid pSSRL, which includes a 1.2-kilobase pair fragment containing the rat liver MAT coding region (15Mingorance J. Alvarez L. Sánchez-Góngora E. Mato J.M. Pajares M.A. Biochem. J. 1996; 315: 761-766Crossref PubMed Scopus (54) Google Scholar). The mutants were identified by sequencing of the complete MAT cDNA. Expression and purification of WT (wild type) and mutant proteins were carried out as described previously (15Mingorance J. Alvarez L. Sánchez-Góngora E. Mato J.M. Pajares M.A. Biochem. J. 1996; 315: 761-766Crossref PubMed Scopus (54) Google Scholar). Protein purity was greater than 95% in all preparations as determined by SDS-polyacrylamide gel electrophoresis. Protein concentration was measured using the Bio-Rad protein assay kit based on the Bradford assay (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). The recombinant WT and MAT mutants analyzed presented similar values of specific activity: 4.36 ± 0.28, 4.19 ± 0.72, 3.97 ± 0.91, 4.22 ± 0.31, 3.89 ± 0.74, 4.23 ± 0.41, 4.89 ± 0.52, 4.01 ± 0.67, and 4.12 ± 0.37 nmol/min/mg protein for WT, C121S, D355S, D355E, R357S, R363S, R363K, G359S, and R357S/R363S, respectively. To exclude that mutant proteins presented any major alteration in their tertiary structure, fluorescence analysis was performed using a Perkin-Elmer were and in 5 pH with at Protein concentration was were the of recombinant WT and MAT mutants were was measured according to the procedure described by et al. (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). of purified recombinant MAT were incubated withS-nitrosoglutathione at 37 °C for min. The was by proteins were incubated with and the of NO was measured by the from reaction with using a nitric oxide were obtained with of MAT activity was measured as described by et al. C. J. J. Biochem. PubMed Scopus Google Scholar) using of the 5 methionine and 5 in the absence of any Samples of 2 × 106 as were washed twice in phosphate/saline buffer and homogenized by and in and pH containing and was obtained by at × g at 4 °C. EGSNO was by chromatography on a column (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), and the enzyme activity was Samples of purified WT and mutant recombinant rat liver MAT were incubated for min at 37 °C in the presence or absence of or or was by chromatography (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), and the enzyme activity was and EGSNO were according to et al. (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). of an of a or in and m pH were The resulting was incubated at for 5 min and then with synthesis was carried out by the described by Beckman et al. J.S. J. 1994; PubMed Scopus Google Scholar) modified by et B. Biochem. 1997; PubMed Scopus Google Scholar). Only of GSNO, EGSNO, and were used. nitrite and nitrate were to NO by incubation with containing (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). The resulting NO was measured by the from reaction with using a nitric oxide (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). The effect of NO on the content of AdoMet was in isolated rat of rat hepatocytes with the cell-permeable NO donor EGSNO induced a reduction of the cellular AdoMet content 15 min of the of EGSNO to the incubation the content of AdoMet about from ± of in control hepatocytes to ± of in hepatocytes with the NO donor The of the of AdoMet by EGSNO in isolated rat hepatocytes was by the NO donor from the incubation medium and the in buffer without EGSNO 15 min of the of the NO donor and of the in medium without EGSNO, cellular AdoMet content to that of hepatocytes hepatocytes were in the presence of the NO donor, the of the cellular content of AdoMet was the next 15 min previously demonstrated (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), incubation with EGSNO 15 min induced the inactivation of MAT ± of protein in control hepatocytes to ± of protein in the presence of 5 Removal of the NO donor from the incubation medium MAT activity to values similar to of the hepatocytes ± of 15 min. We next analyzed whether the effect of EGSNO on AdoMet content in hepatocytes was The of EGSNO to isolated rat hepatocytes induced a of NOx, which to a of AdoMet content reduction of the AdoMet content in hepatocytes was at EGSNO, a where about The of EGSNO to isolated rat hepatocytes as described previously (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), a inactivation of MAT activity not of recombinant WT rat liver enzyme and mutants of MAT was measured after reaction with of In WT rat liver, MAT incubation for min with 100 the S-nitrosylation of about with also induced the inactivation of the shown in is a inverse the of S-nitrosylation and of activity induced by of of of MAT to about inactivation of and inactivation were obtained min of incubation with not We have previously shown (8Avila M.A. Mingorance J. Martı́nez-Chantar M.L. Casado M. Martı́n-Sanz P. Boscá L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar) that of cysteine 121 by serine to as the ability of NO donors to inactivate hepatic incubation of with in the of only about of of MAT The of protein S-nitrosylation has been recently proposed (11Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar) to an acid-base-catalyzed exchange reaction, where the target cysteine residue is located next to basic and amino acids. In human and rat liver MAT, cysteine 121 is not by and basic amino acids by and M. J. Biochem. PubMed Scopus Google Scholar, L. M. Corrales F. Pajares M.A. Mato J.M. PubMed Scopus Google Scholar, L. Corrales F. A. Mato J.M. Biochem. J. 1993; PubMed Scopus Google Scholar). the of liver MAT is one residue of and two of arginine are all found in the vicinity of the group of cysteine 121 Replacement of by serine markedly of the mutant enzyme in the presence of Replacement of arginine 357 or of arginine by serine also to a marked reduction of of the mutant enzyme as with the WT liver MAT Further, a where arginine 357 and arginine were both replaced by serine only about of of MAT after incubation with 100 we have replaced arginine by and by and that these effect on the S-nitrosylation of the mutant by as with the WT liver the results obtained with the mutant enzyme similar results were with the mutant enzyme not an control we have a residue that is by from the group of cysteine to serine and that this effect on the S-nitrosylation of the mutant enzyme by not These that the on MAT S-nitrosylation of arginine arginine or to serine are to of and not to in the of cysteine 121. to the WT recombinant MAT, incubation of mutants D355S, D355E, R357S, R363S, R363K, G359S, and R357S/R363S with of in the of about of of MAT that all cysteine in the protein L. M. Corrales F. Pajares M.A. Mato J.M. PubMed Scopus Google Scholar) were of MAT S-nitrosylation and of MAT purified from E. with WT mutant was incubated in the presence of of for min at 37 °C. The enzyme activity and the of were then determined as described MAT activity is expressed as the of activity after with are the ± of enzyme preparations of rat liver MAT The structural of the of cysteine 121 is based on a for the rat liver MAT previously E. F. Mingorance J. Corrales F.J. Mato J.M. J. 1997; PubMed Scopus Google Scholar) using the for the of E. MAT F. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Biochemistry. 1996; PubMed Scopus (104) Google Scholar). was with Scholar). The of cysteine 121 arginine 357 arginine and are to this the the group of cysteine 121 and the of arginine 357 and arginine are and respectively. The the group of cysteine 121 and the of is We next analyzed whether these mutants were to enzyme incubation of the WT recombinant enzyme with to reduction of the enzyme activity, of cysteine 121 by a serine residue the ability of to inactivate liver MAT Replacement of arginine or arginine by serine to a marked reduction of the ability of to inactivate liver MAT Further, of both arginine 357 and arginine by serine was as as the of cysteine 121 by serine to MAT inactivation the of arginine by by acid, or by serine not the ability of to inactivate liver the results obtained with the mutant enzyme similar results were with the mutant and not of WT liver MAT for min with also induced the inactivation of the enzyme incubation of WT liver MAT with not in the S-nitrosylation of the enzyme not of cysteine 121 by serine the ability of to inactivate liver MAT, of or arginine by or of both arginine 357 and arginine by serine effect on the inactivation of the enzyme by These results indicate that arginine arginine and are for of cysteine these are for the interaction of this with Moreover, these results that in these mutants cysteine 121 is not in the results indicate that NO or related AdoMet content in hepatocytes via and inactivation of hepatic in the hepatic levels of NO induces a rapid S-nitrosylation and inactivation of hepatic MAT (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar), which leads to a rapid of the AdoMet the of the NO leads to the rapid denitrosylation and of hepatic MAT (10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar) and to the rapid of the AdoMet agrees with the that the half-life of hepatic AdoMet is only about 5 min (6Finkelstein J.D. J. Nutr. Biochem. 1990; 1: 228-237Crossref PubMed Scopus (1076) Google Scholar). Further, results indicate that a in the levels of NO, as incubation with EGSNO, is to have a effect on AdoMet We have previously M.A. Carretero M.V. Rodrı́guez E.N. Mato J.M. Gastroenterology. 1998; 114: 364-371Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) that in rat hepatocytes levels MAT is we also found that in rat hepatocytes induced the expression of NO and that the inactivation of MAT was by the NO methyl ester M.A. Carretero M.V. Rodrı́guez E.N. Mato J.M. Gastroenterology. 1998; 114: 364-371Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). levels of NO or related to the abnormal metabolism of methionine in with liver hepatic MAT activity, and content of liver AdoMet have been in with liver cirrhosis and/or in experimental models of liver injury Scopus Google Scholar, J.M. Gastroenterology. Full Text PDF PubMed Scopus Google Scholar, A. Ortiz P. C. Mato J.M. Hepatology. 1998; Scopus Google Scholar, C. I.M. N. M.A. Hepatology. 1990; PubMed Scopus Google Scholar, F. A. Alvarez L. J. Pajares M.A. A. Mato J.M. J. Hepatology. PubMed Scopus Google Scholar, E. M. M. Alvarez L. J. L. Mato J.M. Hepatology. PubMed Google Scholar). In with liver cirrhosis and in experimental models of liver disorders levels of are Geller D.A. J.R. Arias I.M. Boyer J.L. Fausto N. Schaachter D.A. The Liver Biology and Pathobiology. Raven Press, Ltd., New York1994: Scholar, C. A. J. F. M. Hepatology. 1993; 18: PubMed Scopus Google Scholar). The levels in with liver cirrhosis have been reported to to in control to in C. A. J. F. M. Hepatology. 1993; 18: PubMed Scopus Google Scholar). is not be the concentration of in the liver, the results indicate that inactivate liver MAT by about These are for a of AdoMet metabolism by NO. NO many by S-nitrosylation of active site and J.S. 1994; Full Text PDF PubMed Scopus Google Scholar). It is not structural factors govern the of a specific protein cysteine residue by NO. acid-base-catalyzed exchange reaction, where the target cysteine residue is located next to basic and amino has been recently proposed (11Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). The inactivation of hepatic MAT to occur of the residue of cysteine 121 of the enzyme (8Avila M.A. Mingorance J. Martı́nez-Chantar M.L. Casado M. Martı́n-Sanz P. Boscá L. Mato J.M. Hepatology. 1997; 25: 391-396PubMed Google Scholar, 10Ruiz F. Corrales F.J. Miqueo C. Mato J.M. Hepatology. 1998; 28: 1051-1057Crossref PubMed Scopus (104) Google Scholar). results indicate that the of cysteine 121 by serine MAT S-nitrosylation and inactivation by we show that is a inverse the of S-nitrosylation and of 121 is localized at a over the active site of MAT E. F. Mingorance J. Corrales F.J. Mato J.M. J. 1997; PubMed Scopus Google Scholar, F. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. Biochemistry. 1996; PubMed Scopus (104) Google Scholar). two and and has been proposed that in the the of the to the active site F. Biochemistry. 1996; PubMed Scopus (104) Google Scholar). 121 is not for activity the of this amino residue by serine has effect on MAT activity (15Mingorance J. Alvarez L. Sánchez-Góngora E. Mato J.M. Pajares M.A. Biochem. J. 1996; 315: 761-766Crossref PubMed Scopus (54) Google Scholar). The results that of a group in cysteine 121 induces a in the the active site of the enzyme for the by the into the Replacement of the acidic amino or of any of the two basic amino acids localized in the of cysteine 121 by serine markedly the of and inactivate liver Further, results indicate that MAT S-nitrosylation and inactivation are in a mutant enzyme where arginine 357 and arginine were both replaced by the not of (arginine by and by these effect on the S-nitrosylation and inactivation of the mutant by results also indicate that the of a amino in the vicinity of cysteine 121 by serine not S-nitrosylation and inactivation of the mutant enzyme by We also show that MAT inactivation involves the reaction of this with cysteine 121. In this of arginine arginine or by serine not MAT These results indicate that the MAT S-nitrosylation and inactivation following of acidic and basic in the vicinity of cysteine 121 with serine are of of and are not of structural that the or the One of these is that the of the of arginine 357 and arginine be to the of the group of cysteine 121 the of cysteine 121 and the of The of the group of be to the of the of NO These results the experimental as proposed by et al. (11Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar), the S-nitrosylation of protein is by the basic and acidic amino acids surrounding the target Recognition of this topology is likely to prove useful in identifying new targets of protein S-nitrosylation. We and for the synthesis of