Characterization of two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln).
Two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln) were expressed in pyruvate dehydrogenase complex-deletion mutant Escherichia coli JRG1342. The expressed mutant E3s were purified to near homogeneity using DEAE-Sephacel and hydroxyapatite columns. The initial velocity measurements in the absence of products for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines in double-reciprocal plots, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its Km for dihydrolipoamide was dramatically increased by 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on double-reciprocal plots. Its specific activity decreased to 28% of that of wild-type enzyme. Its Km for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in human E3 reaction, is critical to human E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.
Kim
H
H
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Patel
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005822:Genetic Vectors; D005971:Glutamates; D018698:Glutamic Acid; D005973:Glutamine; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005973:Glutamine; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D006639:Histidine; D008058:Dihydrolipoamide Dehydrogenase