Studies of the substrate specificity of cholesterol oxidase from Nocardia erythropolis in the oxidation of 3-hydroxy steroids.

going reduction. However, the carbonyl group of the larger, and more polar, 178-0(3-carboxypropanoyl)glycollyl side chain was not reduced. A 17/3-carboxyl or 178-methoxycarbonyl side chain was not reduced, probably for electronic reasons. In this respect, the reducing power of cortisone reductase resembled that of NaBH4 or lithium tri-t-butoxyaluminium hydride, and was less than that of LiAIH4. Estimates of the binding of various compounds were obtained from inhibition studies. From these it appeared likely that interactions between the enzyme-NADH complex and the 20-0x0 group contribute little to the stability of the ternary enzyme-NADH20-0x0 steroid complex. On the other hand, the 20a-hydroxy configuration (instead of the 20jLhydroxy configuration) evidently generates repulsive forces sufficient to offset the forces that normally bind the rest of the steroid molecule to the enzyme-NAD+ complex. The presence of a hydroxyl group on either of the carbon atoms (C-17 and C-21) adjacent to the reaction site (C-20) did not prevent the enzyme and NAD+ from abstracting the 20a-hydrogen, but in the presence of a hydroxyl group on both of these atoms, this reaction was undetectable. The joint presence of these neighbouring hydroxyl groups would be expected on electronic grounds to make hydride abstraction more difficult, but other enzymes (EC 1.1.1.6 and 1.1.1.94) catalyse the analogous oxidation of the 2-hydroxyl group of glycerol or glycerol phosphate.