Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3α-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis

A human liver 3α-hydroxysteroid dehydrogenase isoenzyme, a member of the aldoŐketo reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the K_m values for both NADP(H) and substrates. Here we report kinetic alterations in binding of the coenzymes and the activator to the enzyme caused by site-directed mutagenesis of Lys-270 and Arg-276, which are strictly conserved among the aldoŐketo reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the K_m for NADP⁺ of 22- and 290-fold respectively; the K_m for alcohol substrate and the k_cat of the NADP⁺-linked reaction were also elevated, by 9- and 5-fold respectively. No kinetic constant of the NAD⁺-linked reaction was altered by more than 3-fold. Calculation of the free-energy changes showed that the 2ƀ-phosphate group of NADP⁺ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wild-type enzyme, and the mutagenesis to K270M and R276M destabilized the binding energy of NADP⁺ by 6.3 and 13.0 kJ/mol (1.5 and 3.1 kcal/mol) respectively. In addition, the mutations attenuated enzyme activation by sulphobromophthalein, which bound to the mutant enzymes as an inhibitor. The inhibition for the R276M mutant was competitive with respect to NADP⁺ and non-competitive with respect to the substrate, whereas that for the K270M mutant was mixed-type, showing activation at coenzyme concentrations greater than 20ȕK_m. These results suggest that the two basic residues in the 3α-hydroxysteroid dehydrogenase isoenzyme play crucial roles in binding both the negatively charged 2ƀ-phosphate group of NADP⁺ and the sulphonic groups of sulphobromophthalein.