NADP.sup.+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

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...

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Published in:PloS one 2012-11, Vol.7 (11), p.e50329
Main Authors: González, Beatriz, Garrido, Francisco, Ortega, Rebeca, Martínez-Júlvez, Marta, Revilla-Guarinos, Ainhoa, Pérez-Pertejo, Yolanda, Velázquez-Campoy, Adrián, Sanz-Aparicio, Julia, Pajares, María A
Format: Article
Language:English
Subjects:
Amino acids
Calorimetry
Enzymes
Oligomers
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Summary: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.sup.+ with a 1:1 stoichiometry, the cofactor enhancing [beta] to [alpha]2-dimer binding affinity. Mutants lacking residues involved in NADP.sup.+ 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0050329