title: Thermodynamic analysis of subunit interactions in Escherichia coli molybdopterin synthase.
abstract: The molybdopterin (MPT) synthase complex in Escherichia coli consists of two MoaE subunits and two MoaD subunits in a heterotetrameric structure with the two MoaE subunits forming a central dimer. Each MoaD subunit binds to a single MoaE molecule to form two identical MoaE/MoaD interfaces. Here we define the thermodynamic properties of the interaction between MoaE and MoaD in MPT synthase using a H/D exchange and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy based method termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX-derived protein folding free energies and m values are reported for MoaE in the presence and absence of MoaD and MoaD-SH, the thiocarboxylated form of MoaD that is essential for the catalytic activity of MPT synthase. The protein folding free energy measurements were used to calculate a dissociation constant of 17 +/- 7 microM for the binding of MoaD to MoaE in inactive MPT synthase and a dissociation constant of 2.6 +/- 0.9 microM for the binding of MoaD-SH to MoaE in active MPT synthase. The increased binding affinity of MoaD-SH for MoaE is consistent with a previously proposed mechanism for the MPT synthase reaction. Using the increased m values exhibited by MoaE in the presence of either MoaD subunit, the solvent accessible surface area buried upon formation of the subunit interface in MPT synthase was estimated to be 2378 A(2) for inactive MPT synthase and 4117 A(2) for active MPT synthase.
authors.affiliation: Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Tong
authors.forename: Margot M
authors.initials: MM
authors.lastname: Wuebbens
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D003067:Coenzymes; D041961:Deuterium Exchange Measurement; D019281:Dimerization; D004789:Enzyme Activation; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008667:Metalloproteins; D008956:Models, Chemical; D000090006:Molybdenum Cofactors; D009942:Organometallic Compounds / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D011621:Pteridines / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013466:Sulfurtransferases / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D003067:Coenzymes; D029968:Escherichia coli Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009942:Organometallic Compounds; D021122:Protein Subunits; D011621:Pteridines; D013466:Sulfurtransferases; C022261:molybdopterin; C085585:molybdopterin synthase