Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3 alpha-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis.
A human liver 3 alpha-hydroxysteroid dehydrogenase isoenzyme, a member of the aldo-keto reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the Km 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 aldo-keto reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the Km for NADP+ of 22- and 290-fold respectively; the Km for alcohol substrate and the kca1 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 2'-phosphate group of NADP+ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wildtype 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 20 x Km. These results suggest that the two basic residues in the 3 alpha-hydroxysteroid dehydrogenase isoenzyme play crucial roles in binding both the negatively charged 2'-phosphate group of NADP+ and the sulphonic groups of sulphobromophthalein.
Matsuura
K
K
Biochemistry Laboratory, Gifu Pharmaceutical University, Japan.
Tamada
Y
Y
Sato
K
K
Iwasa
H
H
Miwa
G
G
Deyashiki
Y
Y
Hara
A
A
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000235:genetics* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000235:genetics / Q000378:metabolism*; D013448:Sulfobromophthalein / Q000494:pharmacology*
D013448:Sulfobromophthalein; D009249:NADP; D001120:Arginine; D015096:3-Hydroxysteroid Dehydrogenases; D008239:Lysine