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.