Lung Carbonyl Reductase Research Articles

Lysine residues in the coenzyme-binding region of mouse lung carbonyl reductase.

Tetrameric carbonyl reductase (CR, EC 1.1.1.184) of guinea-pig, mouse and pig lung differs from CRs of other mammalian tissues in subunit structure, broad substrate specificity for aromatic and aliphatic carbonyl compounds, reversibility of the reaction and sensitivity to pyrazole (Nakayama et al., 1982, 1986; Oritani et al., 1992). It is also uniquely activated by fatty acids and dipyridyl compounds (Hara et al., 1992a, 1993). The cDNA for pig lung has been cloned (Nakanishi et al., 1993). The enzyme is composed of 244 amino acids, and is structurally related to members of the short-chain alcohol dehydrogenase (SCAD) family, which includes eucaryotic and procaryotic enzymes with different substrate specificity (Persson et al., 1990; Neidle et al, 1992; Krozowski, 1992).

Purification and characterization of pig lung carbonyl reductase

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a M r of 103,000, consisting of apparent identical subunits of M r 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P) +. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro- S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5α- and 5β-androstanes, and the respective reduced products were identified as 3β- and 3α-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [ S] 0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.

Activation of carbonyl reductase from pig lung by fatty acids

The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing K a values of 2–14 μ m which were lower than the values of 21–125 μ m for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the K a values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both V max and [ S] 0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 μ m. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase.

Kinetic mechanism of pulmonary carbonyl reductase.

The kinetic mechanism of guinea-pig lung carbonyl reductase was studied at pH 7 in the forward reaction with five carbonyl substrates and NAD(P)H and in the reverse reaction with propan-2-ol and NAD(P)+. In each case the enzyme mechanism was sequential, and product-inhibition studies were consistent with a di-iso ordered bi bi mechanism, in which NAD(P)H binds to the enzyme first and NAD(P)+ leaves last and the binding of cofactor induces isomerization. The kinetic and binding studies of the cofactors and several inhibitors such as pyrazole, benzoic acid, Cibacron Blue and benzamide indicate that the cofactor and Cibacron Blue bind to the free enzyme whereas the other inhibitors bind to the binary and/or ternary complexes.