Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1-prostaglandin D2 synthase activity in the absence of NADP(H)

Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F-2 alpha (PGF(2 alpha)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2 alpha). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP+. Both PGD(2) and PGF(2 alpha) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP+ or after heat treatment of these enzymes at 100 degrees C for 5 min. The K-m, V-max, pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 mu m, 151 and 57 nmol center dot min-1 center dot(mg protein)-1, 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2 alpha) synthase activity were 29 and 33 mu m, 169 and 240 nmol center dot min-1 center dot(mg protein)-1, 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2 alpha) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2 alpha) in the absence of NADPH or NADP+ and in the presence of NADPH, respectively.