Kinetic alteration of a human dihydrodiol/3α-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine

Abstract

Human dihydrodiol dehydrogenase with 3α-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1Ő1C4) that belong to the aldoŐketo reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1Ő1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the Km for NADP+ and differently influenced the Km and kcat for substrates depending on their structures. The kinetic constants for bile acids with a 12α-hydroxy group were decreased 1.5Ő7-fold and those for the other substrates were increased 1.3Ő9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17β-oestradiol, phenolphthalein and flufenamic acid and 3,5,3´,5´-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the Km for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.