title: Asparagine-473 residue is important to the efficient function of human dihydrolipoamide dehydrogenase.
abstract: Dihydrolipoamide dehydrogenase (E3) 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. His-457 of Pseudomonas putida E3 is suggested to interact with the hydroxyl group of Tyr-18 of the other subunit and with Glu-446, a component in the last helical structure. To examine the importance of the suggested interactions in human E3 function, the corresponding residue of human E3, Asn-473, was substituted to Leu using site-directed mutagenesis. The E3 mutant was expressed in Escherichia coli and highly purified using an affinity column. Its E3 activity was decreased about 37-fold, indicating that Asn-473 residue was important to the efficient catalytic function of human E3. Its slightly altered spectroscopic properties implied that small conformational changes could occur in the E3 mutant.
authors.affiliation: Department of Chemistry, College of Natural Science, Daegu University, Kyoungsan 712-714, Korea. hjkim@daegu.ac.kr
authors.forename: Hakjung
authors.initials: H
authors.lastname: Kim
mesh_terms: D001216:Asparagine / Q000737:chemistry* / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D001216:Asparagine; D008058:Dihydrolipoamide Dehydrogenase