Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase.
Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.
Steffan
J S
JS
Department of Biological Chemistry, College of Medicine, University of California, Irvine 92717.
McAlister-Henn
L
L
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D002850:Chromatography, Gel; D003062:Codon; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D000085:Acetates; D003062:Codon; D046911:Macromolecular Substances; D001224:Aspartic Acid; D006639:Histidine; D001216:Asparagine; D008291:Malate Dehydrogenase; D007930:Leucine; D019342:Acetic Acid