Folding of dimeric methionine adenosyltransferase III: identification of two folding intermediates.
Methionine adenosyl transferase (MAT) is an essential enzyme that synthesizes AdoMet. The liver-specific MAT isoform, MAT III, is a homodimer of a 43.7-kDa subunit that organizes in three nonsequential alpha-beta domains. Although MAT III structure has been recently resolved, little is known about its folding mechanism. Equilibrium unfolding and refolding of MAT III, and the monomeric mutant R265H, have been monitored using different physical parameters. Tryptophanyl fluorescence showed a three-state folding mechanism. The first unfolding step was a folding/association process as indicated by its dependence on protein concentration. The monomeric folding intermediate produced was the predominant species between 1.5 and 3 m urea. It had a relatively compact conformation with tryptophan residues and hydrophobic surfaces occluded from the solvent, although its N-terminal region may be very unstructured. The second unfolding step monitored the denaturation of the intermediate. Refolding of the intermediate showed first order kinetics, indicating the presence of a kinetic intermediate within the folding/association transition. Its presence was confirmed by measuring the 1,8-anilinonaphtalene-8-sulfonic acid binding in the presence of tripolyphosphate. We propose that the folding rate-limiting step is the formation of an intermediate, probably a structured monomer with exposed hydrophobic surfaces, that rapidly associates to form dimeric MAT III.
Sánchez del Pino
Manuel M
MM
Division of Hepatology and Gene Therapy, University of Navarra, Irunlarrea 1, 31008 Pamplona, Spain. mspino@unav.es
Pérez-Mato
Isabel
I
Sanz
Jesús M
JM
Mato
José M
JM
Corrales
Fernando J
FJ
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D008716:Methionine Adenosyltransferase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D006639:Histidine; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D008716:Methionine Adenosyltransferase