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Identification and characterization of dihydrolipoamide dehydrogenase deficiency

by Liu, Te-Chung

Abstract (Summary)
This study involved identification of two missense mutations in an E3-deficient patient, characterization of the kinetic and physical properties of these two missense E3 proteins and two other active base missense mutant proteins which were created by site-directed mutagenesis. Fibroblasts cultured from the patient contained about 6% of the E3 activity of cells from a normal subject. Western and Northern blot analyses indicated that the patient's cells had a reduced amount of protein but normal amounts of E3 mRNA when compared to those of control cells. Direct sequencing of E3 cDNA derived from the patient's RNA as well as each subclone of the cDNA revealed that the patient had two substitution mutations in the E3 coding region. One identified mutation changed a single nucleotide from A to G, resulting in the substitution of E (GAA) for K-37(AAA) (E3 K-37 to E). The other point mutation was a nucleotide change from C to T, resulting in the substitution of L (CTG) for P-453 (CCG) (E3 P-453 to L). The specific activity of purified recombinant E3 K-37 to E was decreased to 50% and recombinant E3 P-453 to L was decreased to 0.04% when compared to the wild type E3. Kinetic studies of the E3 K-37 to E showed that the reac tion mechanism of this mutant protein followed the 'Ping Pong' mechanism. The Km for dihydrolipoamide and NAD^+ remain the same as E3, however the Kcat decreased to 50% when compared to E3. The chemical and physical properties of E3 K-37 to E and E3 P-453 to L together with E3 E-452 to Q and E3 H-457 to Q were analyzed. Molecular sieving showed that the dimerization of E3 K-37 to E, E3 E-457 to Q, and E3 H-452 to Q was not affected by the mutation, while only 50% of E3 P-453 to L was in the dimer form. Quantitation studies of FAD showed that the amount of FAD of the E3 E-457 to Q, and E3 H-452 to Q decreased by 10%, E3 K-37 to E decreased by 30% while E3 P-453 to L decreased by 70%. UV-visible, Fluorescence and CD spectral represented the lower amount of FAD of E3 K-37 to E, and E3 P-453 to L. The E3 H-452 to Q showed slow reduction of FAD without formation of NADH. The E3 E-457 to Q showed that the smaller peak at 510 nm and the formation of NADH at peak 460 nm was maintained with a slow decreasing rate. The CD spectra also showed that the four mutations decreased the ?-helix and ?-sheet content and caused the conformation change of the E3 mutant protein.
Bibliographical Information:

Advisor:

School:Case Western Reserve University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:dihydrolipoamide dehydrogenase deficiency

ISBN:

Date of Publication:01/01/1994

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