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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/61468
Title: NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer
Authors: González, Beatriz; Garrido, Francisco; Ortega, Rebeca; Revilla-Guarinos, Ainhoa; Sanz-Aparicio, J.; Pajares, María A.
Keywords: Methionine adenosyltransferase
NADP binding site
Binding affinity
Issue Date: 29-Nov-2012
Abstract: 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 β subunits and catalytic α2 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 α2 and β 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 α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-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 β 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 β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed on the light of the recent identification of several splicing forms of this subunit in hepatoma cells.
Description: This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.
Publisher version (URL): http://dx.doi.org/10.1371/journal.pone.0050329
URI: http://hdl.handle.net/10261/61468
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0050329
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