title: Amide hydrogen exchange shows that malate dehydrogenase is a folded monomer at pH 5.
abstract: Although there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH-induced changes in solvent accessibility, MDH was exposed to D(2)O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the intrinsic rate of hydrogen exchange, large increases in deuterium levels were found at pH 5 only in peptic fragments derived from the subunit binding surface of MDH. In parallel experiments, elevated deuterium levels were also found in the same regions of MDH monomer trapped inside a mutant form of the chaperonin GROEL: This selective increase in hydrogen exchange rates, which was attributed to increased solvent accessibility of these regions, provides new evidence that MDH is a monomer at pH 5.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: D L
authors.initials: DL
authors.lastname: Smith
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
chemical_list: D000577:Amides; D018834:Chaperonin 60; D010446:Peptide Fragments; D021122:Protein Subunits; D003903:Deuterium; D008291:Malate Dehydrogenase; D010434:Pepsin A