RESUMEN
Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/química , Dihidrolipoamida Deshidrogenasa/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Cartilla de ADN/genética , Dihidrolipoamida Deshidrogenasa/deficiencia , Dihidrolipoamida Deshidrogenasa/metabolismo , Dimerización , Flavina-Adenina Dinucleótido/análisis , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , NAD/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the K(cat) of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.
