Kinetic mechanism of porcine testicular 17 beta-hydroxysteroid dehydrogenase.
A study on product inhibition of 17 beta-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP+ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP+, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP+, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP+ and testosterone. The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17 beta-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex. From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D008297:Male; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013552:Swine; D013737:Testis / Q000201:enzymology*; D013739:Testosterone / Q000378:metabolism
D013739:Testosterone; D000735:Androstenedione; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases