Activity of human dihydrolipoamide dehydrogenase is largely reduced by mutation at isoleucine-51 to alanine.
Dihydrolipoamide dehydrogenase (E3) belongs to the pyridine nucleotide-disulfide oxidoreductase family including glutathione reductase and thioredoxin reductase. It catalyzes the reoxidation of dihydrolipoyl moiety of the acyltransferase components of three alpha-keto acid dehydrogenase complexes and of the hydrogen-carrier protein of the glycine cleavage system. Isoleucine-51 of human E3, located near the active disulfide center Cys residues, is highly conserved in most E3s from several sources. To examine the importance of this highly conserved Ile-51 in human E3 function, it was substituted with Ala using site-directed mutagenesis. The mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 100-fold, indicating that the conservation of the Ile-51 residue in human E3 was very important to the efficient catalytic function of the enzyme. Its altered spectroscopic properties implied that conformational changes could occur in the mutant.
Kim
Hakjung
H
Department of Chemistry, College of Natural Science, Daegu University, Kyoungsan 712-714, Korea. hjkim@daegu.ac.kr
D000409:Alanine / Q000235:genetics*; D019943:Amino Acid Substitution; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007532:Isoleucine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation
D007532:Isoleucine; D008058:Dihydrolipoamide Dehydrogenase; D000409:Alanine