The role of cysteine-150 in the structure and activity of rat liver S-adenosyl-l-methionine synthetase

Abstract

The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms.

Role of an Intrasubunit Disulfide in the Association State of the Cytosolic Homo-oligomer Methionine Adenosyltransferase

“…Determination of the Free Sulfhydryl Content and Location of the Disulfide Bond-The number of free ϪSH groups for wild type and mutant MATs was determined as described previously using NEM labeling (15,22). In addition, for quantitation of free cysteine residues and disulfide bonds in wild type and mutant MAT proteins, samples of the purified proteins were dialyzed against ammonium acetate extensively and lyophilized, and the content of reduced and oxidized cysteine residues was determined by mass spectrometry.…”

“…This is of special relevance if we take into account that the product of MAT reaction is S-adenosylmethionine, the main methyl donor for the transmethylation reactions (18). This enzyme presents 10 cysteine residues/subunit (19,20), and under several in vitro and in vivo conditions it has been possible to demonstrate their role on the activity and oligomeric state: (a) inactivation and dissociation of MAT I is produced by N-ethylmaleimide (NEM) modification of two ϪSH groups (21,22); (b) site-directed mutagenesis of Cys 69 renders the enzyme mainly as dimers (23); (c) all of the mutants on the cysteines comprised between residues 35 and 105 have some effect on the MAT I/MAT III ratio (23); (d) the enzyme activity is inhibited by GSSG (2) and nitrosylation of Cys 121 (24,25); (e) inhibition of the glutathione synthesis leading to a 30% reduction in the GSH levels, and hence to the alteration of the GSH/GSSG ratio, correlates with a decrease in MAT activity (26); and (f) low MAT activity and mainly dimers are detectable under the oxidative conditions of alcohol liver cirrhosis (27). Moreover, a disulfide bond between Cys 35 and Cys 61 that are located in the ␤-sheet of contact between dimers, according to the crystal structure, has been identified (15,28).…”

Analysis of the 5′ non‐coding region of rat liver S‐adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme

“…Due to the known importance of AdoMet synthetase sulfbydryl groups [14,15] in maintaining the enzyme structure and activity, the high-1W; form of the enzyme was purified as described in [3], to then be chromatographed on a thiopropyl-sepharose column. After its elution with a cysteine gradient, AdoMet synthetase activity was determined (data not shown), the active fractions pooled and a sample loaded on a 10% SDS-PACE gel under reducing conditions.…”

“…3), a result that is consistent with the M, deduced from the cDNA sequence. The reason for the modification in the electrophoretic be- haviour of the enzyme afte= its chromatography on the thiopropyl-sepharose column is not clear, but might be related to the role of sulfhydryl groups in maintaining the activity and structure of the enzyme [14,15].…”

Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP

“…equilibrated in 0.1 % TFA in water. The chromatography was carried out at a flow rate of 1 ml/min, as described in [14], with a three-step gradient involving 0.1 % TFA in water (solvent A) and 0.1 % TFA in 50 % acetonitrile (solvent B). The absorbance at 214 nm was monitored, and 1 ml fractions were collected and subjected to Cerenkov counting for detection of the 32P labelling.…”