NADP(+) Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer
Por:
Gonzalez, B, Garrido, F, Ortega, R, Martinez-Julvez, M, Revilla-Guarinos, A, Perez-Pertejo, Y, Velazquez-Campoy, A, Sanz-Aparicio, J and Pajares, M
Publicada:
26 nov 2012
Resumen:
Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory beta subunits and catalytic alpha 2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant alpha 2 and beta subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with alpha 2 dimers associated to single beta subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing beta to alpha 2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the beta subunit were able to oligomerize with alpha 2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the beta subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in beta to alpha 2-dimer interaction. Finally, the implications that the presence of different N-terminals in the beta subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.
Filiaciones:
Gonzalez, B:
Inst Quim Fis Rocasolano CSIC, Dept Cristalog & Biol Estruct, Madrid, Spain
Garrido, F:
Inst Invest Biomed Alberto Sols CSIC UAM, Madrid, Spain
Ortega, R:
Inst Quim Fis Rocasolano CSIC, Dept Cristalog & Biol Estruct, Madrid, Spain
Martinez-Julvez, M:
Univ Zaragoza, Dept Bioquim & Biol Mol & Celular, Zaragoza, Spain
Inst Biocomputac & Fis Complejos, Unidad Asociada IQFR BIFI, Zaragoza, Spain
:
Inst Quim Fis Rocasolano CSIC, Dept Cristalog & Biol Estruct, Madrid, Spain
Perez-Pertejo, Y:
Univ Leon, Dept Farmacol & Toxicol INTOXCAL, E-24071 Leon, Spain
Velazquez-Campoy, A:
Univ Zaragoza, Dept Bioquim & Biol Mol & Celular, Zaragoza, Spain
Inst Biocomputac & Fis Complejos, Unidad Asociada IQFR BIFI, Zaragoza, Spain
Fdn ARAID, Diputac Gen Aragon, Zaragoza, Spain
Sanz-Aparicio, J:
Inst Quim Fis Rocasolano CSIC, Dept Cristalog & Biol Estruct, Madrid, Spain
Pajares, M:
Inst Invest Biomed Alberto Sols CSIC UAM, Madrid, Spain
IdiPAZ, Mol Hepatol Grp, Madrid, Spain
Green Submitted, Green Published, gold
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