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

Human dihydrodiol dehydrogenase with 3 alpha -hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto red uctase (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 t he coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylal anine 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 K-m for NADP(+) and differently influen ced the K-m and k(cat) for substrates depending on their structures. The ki netic constants for bile acids with a 12 alpha -hydroxy group were decrease d 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive i nhibitors such as hexoestrol analogues, 17 beta -oestradiol, phenolphthalei n 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 incre ased the K-m for the coenzyme and substrate of the mutant enzymes more high ly than those of the wild-type enzyme. These results indicate the importanc e of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coe nzyme, as well as its involvement in the conformational change by binding n on-essential activators.