| Summary: | 碩士 === 中山醫學大學 === 營養科學研究所 === 90 === The Role of T-44,Y-118 and K-131 in the Reaction Mechanism of Human Dihydrolipoamide Dehydrogenase(E3)
Ya-Hui Chen
Department of Nutritional Science,Chungshan Medical University,No.110,Sec.1,Chien-Kuo N.Rd.,Taichung City,Taiwan,R.O.C.
ABSTRACT
Dihydrolipoamide dehydrogenase (E3) belongs to the enzyme family of pyridine nucleotide-disulfide oxidoreductases and catalyzes the trans- fer of electrons from dihydrolipoamide to FAD cofactor , then to the NAD+. E3 is also the common com ponent of α-ketoacid dehydrogenase complexes including pyruvate dehydrogenase (PDC) , branched-chain α-ketoacid dehydrogenas complexes and α-ketoglutarate dehydrogenas complexes. A deficiency in E3 leads to the deficiency of all these three α-ketoacid dehydrogenase complexes. E3 plays an important role in the energy metabolism indeed. To investigate the reaction mechanism of human dihydrolipoamide dehydrogenase (E3), three mutant human E3s, T44S, Y118E and K131R, were overexpressed, purified and characterized. The specific activities of mutant proteins are 28.5%, 37.5% and 31.5 % to that of the wild-type E3. The FAD content analysis indicated that these three mutant E3s have about 71.3 %, 87.9% and 57.7% of FAD content compared to that of wild-type E3. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetics data demonstrat- ed that the kcat of forward reaction of mutant proteins were decreased to about 79.2% (T44S), 50.5% (Y118E) and 31.3% (K131R). The kcat of reverse reactions were decreased to about 32.9% (T44S), 29.8% (Y118E) and 80.9 % (K131R) compared to that of wild-type E3.In T44S and Y118E the Km of DHL and NAD+ were lower than E3. The mild-point oxidation-reduction potential of mutant proteins were increased from -314 mV (E3) to -290 mV (T44S) and -270 mV (Y118E). It suggests T-44, Y-118 and K-131 involved in FAD binding and oxidoreduction of FAD during the reaction.
Keywords: Dihydrolipoamide Dehydrogenase, site-directed mutagenesis, enzyme kinetics.
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