Folding of Dimeric Methionine Adenosyltransferase III

Abstract

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 ␣-␤ 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.

Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases

“…Thus, in this denaturant range the SS alterations take place together with the largest changes in the tryptophan environment, leading to a monomeric intermediate (J), which is the species detected by sedimentation velocity and that is prone to aggregation according to the gel filtration results. All these results together suggest multi-state denaturation for Mj-MAT, although the number and association state of the intermediates differs from those of MAT III and BsMAT [11,12,23]. Furthermore, MATs also differ in the polar character of their intermediates as judged from their ANS binding capacities.…”

“…These data suggest incomplete unfolding of Mj-MAT even at the highest urea concentration tested, although monomer production is obtained according to the s 20,w value. Comparison with the available data for other members of the MAT family revealed that mesophilic isoenzymes, MAT III [11,12] and BsMAT [23], show complete loss of CD signature and total red-shift of their λ max at 8M urea. Moreover, Mj-MAT retains activity even in 6 M urea, whereas complete loss of AdoMet synthesis and PPP i hydrolysis was already observed at 1 M urea with MAT III [11,12] and BsMAT [23].…”

“…Comparison with the available data for other members of the MAT family revealed that mesophilic isoenzymes, MAT III [11,12] and BsMAT [23], show complete loss of CD signature and total red-shift of their λ max at 8M urea. Moreover, Mj-MAT retains activity even in 6 M urea, whereas complete loss of AdoMet synthesis and PPP i hydrolysis was already observed at 1 M urea with MAT III [11,12] and BsMAT [23]. Thus, these results indicate that the differences between Mj-MAT and mesophilic MATs result not only in increased thermostability, but also in higher resistance to chemical denaturation.…”

“…Dissociation of oligomers due to centrifugal forces has been reported for several proteins and depends on the strength of the interactions between subunits [24]. Comparison with MAT III and BsMAT, which already dissociate at <1M urea [11,12,23], indicate that the increased resistance of Mj-MAT to denaturation seems to derive from an enhanced stability of the dimer. The extra stability provided by subunit association seems to be one reason for hyperthermophiles to prefer oligomeric proteins [25].…”

Proteomic analysis of human hepatoma cells expressing methionine adenosyltransferase I/III

“…Although all three MAT forms share 85% of the aminoacidic sequence [18,19], they differ in their kinetic and regulatory properties [14]. While MATI/III are activated by the substrates methionine and ATP [20], MATII is inhibited by the product of the reaction, SAM [17]. Consistently, cells expressing MAT1A have higher SAM levels than cells expressing MAT2A [21].…”