title: Spectroscopic studies of the characterization of recombinant human dihydrolipoamide dehydrogenase and its site-directed mutants.
abstract: In this paper, we report the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human dihydrolipoamide dehydrogenase in Escherichia coli and detailed spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme. One mutation (K37E) has been identified in a patient lacking dihydrolipoamide dehydrogenase activity and has been reported previously (Liu, T.-C., Kim, H., Arizmendi, C., Kitano, A., and Patel, M. S. (1993) Proc. Natl. Acad. Sci. USA. 90, 5186-5190), while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457). Circular dichroic and fluorescence spectroscopic data illustrate the role of these amino acids in maintaining the structure and function of human dihydrolipoamide dehydrogenase. While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant. The K37E mutant shows very little deviation from the wild-type enzyme.
authors.affiliation: Department of Nutrition, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: T C
authors.initials: TC
authors.lastname: Liu
authors.forename: L G
authors.initials: LG
authors.lastname: Korotchkina
authors.forename: S L
authors.initials: SL
authors.lastname: Hyatt
authors.forename: N N
authors.initials: NN
authors.lastname: Vettakkorumakankav
authors.forename: M S
authors.initials: MS
authors.lastname: Patel
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000737:chemistry* / Q000502:physiology; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D008058:Dihydrolipoamide Dehydrogenase