Role of an intrasubunit disulfide in the association state of the cytosolic homo-oligomer methionine adenosyltransferase.
Recombinant rat liver methionine adenosyltransferase has been refolded into fully active tetramers (MAT I) and dimers (MAT III), using as a source chaotrope-solubilized aggregates resulting from specific washes of inclusion bodies. The conditions of refolding, dialysis in the presence of 10 mm dithiothreitol or 10 mm GSH with 1 mm GSSG, allowed the production of both isoforms, the nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mm dithiothreitol yielded mainly MAT III in a concentration-dependent equilibrium with the homotetramer MAT I. However, refolding in the presence of the redox pair GSH/GSSG resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer interconversion has been found related to the production of a single intramolecular disulfide in methionine adenosyltransferase during the GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine mutants as cysteines 35 and 61. In addition, a kinetic intermediate in the MAT I dissociation to MAT III has been detected. The physiological importance of these results is discussed in light of the structural and regulatory data available.
Sanchez-Perez
Gabino F
GF
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain.
Gasset
Maria
M
Calvete
Juan J
JJ
Pajares
Maria A
MA
D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D013058:Mass Spectrometry; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D004220:Disulfides; D005982:Glutathione Transferase; D008716:Methionine Adenosyltransferase; D014357:Trypsin; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol