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Studies on site-specifically modified human dihydrolipoamide dehydrogenase

by Hakjung, Kim

Abstract (Summary)
The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in E. coli using a ? P L promoter-driven prokaryotic expression vector. Since wild-type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli JRG1342. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 possessed an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about 36% of the mature E3 activity. The double reciprocal plot for the mature E3 in the direction of NAD^+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. This study indicated that the mature recombinant human E3 was an active enzyme whose kinetic properties were similar to those of mammalian E3s. Two site-specifically mutated human E3s (His-452 ? Gln and Glu-457 ? Gln) were expressed in E. coli JRG1342. The double reciprocal plot for the Gln-452 mutant E3 in the direction of NAD^+ reduction showed parallel lines, 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 K m for dihydrolipoamide was dramatically increased by about 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-452 mutant E3 possessed about 2-fold higher activity at pH 8.5 than the activity at pH 8.0, indicating that the Gln-452 mutant E3 probably catalyzed the reaction with the help of water molecules (hydroxyl ions) in assay solution. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed the E3 reaction via a sequential mechanism in the direction of NAD^+ reduction based on the intersecting lines shown on a double reciprocal plot. Its specific activity decreased to 28% of that of wild-type enzyme. Its K m 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 E3 reaction, is critical to 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.
Bibliographical Information:

Advisor:

School:Case Western Reserve University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:modified human dihydrolipoamide dehydrogenase

ISBN:

Date of Publication:01/01/1992

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