The enzymic mechanism of the addition and removal of hydrogen at adjacent carbon atoms of steroids is only partially understood. These reactions include the hydrogenation of Δ4- and Δ1-dehydrosteroids, and the introduction of double bonds into the same positions of saturated compounds (Talalay, 1957a).

Δ-Reductases

In animal tissues and in some micro-organisms the Δ4-3-ketone group of steroid hormones undergoes hydro-genation to the A :B trans (5a-H) and cis (5β-H) products. Tom-kins (1956a, 1957) has shown that the soluble component of the livers of various mammalian species contains steroid reductases which in the presence of TPNH (and in some instances also DPNH) reduce the double bonds of various steroids stereo-specifically to yield exclusively the A;B cis product. These enzymes are extremely fastidious in their substrate requirements since apparently discrete catalytic proteins are concerned with the hydrogenation of even such closely related compounds as Cortisol and cortisone. Other enzymes, localized in the liver microsomes, carry out the reduction of the same substrates to yield the A:B trans product (Forchielli and Dorfman, 1956). Separate liver enzymes also carry out the Δ1-reduction of 1-de-hydrosteroids (Tomkins, 1956a). These reactions appear to be all irreversible. Although it has been clearly shown that TPNH can function as the reductant, there is uncertainty in the steroid reductions whether the pyridine nucleotide reacts directly with the substrate, or whether additional hydrogen carriers such as flavines may be involved. A detailed analysis of the pyridine nucleotide catalysed reduction of orotic to L-dihydro-orotic acid has revealed that the reaction is mediated through a flavine group (Graves and Vennesland, 1957; Friedmann and Vennesland, 1958). Although the action of dihydro-orotic dehydrogenase proceeds stereospecifically with respect to the side of the pyridine nucleotide ring (side I), and gives only the L-dihydro-orotate, there is relatively free exchange of the reducing hydrogen with the medium as shown by conducting the reaction in D20, and by the fact that DPND carrying deuterium (on side I or side II) did not transfer deuterium to the product.

Δ-Dehydrogenases

Numerous micro-organisms carry out the removal of hydrogen from adjacent carbon atoms of steroids principally at C(1) and C(4) to yield the Δ1- and Δ4-dchydro-products (Talalay, 1957a; Vischer and Wettstein, 1958). Recently, these reactions have been studied with partially purified, steroid-induced (adaptive) enzyme preparations from Pseudomonas testosteroni (Levy and Talalay, 1957, 1959). The enzymic activities are associated with small nucleic acid-containing intracellular particles. The reactions proceed by a direct dehydrogenation, and do not involve hydroxylated or other oxygenated intermediates. The dye phenazine methosulphate may substitute efficiently for the unknown natural electron acceptor. The reactions are conveniently studied by observing the reduction of the dye spectrophotometrically under anaerobic conditions.

Although the A-reductases have not been purified to the same degree as some of the hydroxysteroid dehydrogenases, it is nevertheless clear that these enzymes exercise chemical and steric specificity. At least two enzymes are concerned in these reactions:

Crude steroid-adapted extracts of Ps. testosteroni can also convert 5β-androstane compounds to the l,4-diene-3-one grouping, whereas this activity is absent from purified Δ1- and Δ4-5a-dehydrogenase preparations. Although no substrate has been available for the specific assay of Δ4-5β-dehydrogenase, the evidence clearly points to the existence of a discrete enzyme which introduces the A*-double bond into Δβ cis compounds. Attempts to reverse A-dehydrogenase reactions have not been successful.

The precise mechanisms of the A-reductase and Δ-dehydrogenase reactions are not clearly understood. Discrete proteins are concerned with hydrogen transfer at positions C(1), C(4) (5a-H) and C(4) (5β-H). This establishes the positional and to some extent the steric specificity of these enzymes. Independent observations have pointed out that the Δ1-dehydrogenase reactions proceed with only the natural enantiomorphs of synthetic racemic aldosterone and racemic cortisone (Vischer, Schmidlin and Wettstein, 1956). It would be of considerable interest to define precisely the steric relationship of the hydrogens undergoing addition or removal in each case. Detailed analysis of some enzymic reactions (e.g. fumarase, aspartase and aconitase) has revealed that the addition and removal of groups to and from adjacent carbon atoms proceeds as a as-oriented process (Fisher et al., 1955; Englard and Colowick, 1955, 1956, 1957; Farrar et al., 1957; Englard, 1958; Krasna, 1958). If the addition and removal of hydrogens to and from steroids are stereospecific processes, and if they occur in a cis-directed manner, in analogy with other enzyme reactions, it may be predicted that reactions at C(4)-(5) will involve either the 4a, 5a-hydrogens or the 4, 5β-hydrogens, depending upon the A ; B ring fusion. No basis exists for distinguishing whether the steric course at C(1)-(2) involves la, 2a- or 1β, 2β-hydrogens.

Preliminary studies have been carried out with partially purified Δ1-dehydrogenase of Ps. testosteroni to delineate the substrate specificity of this enzyme (Levy and Talalay, 1959). A 3-ketone group is essential for enzymic activity. Examination of the effects on reaction velocity of structural modifications at other points of the molecule has revealed that the areas of enzyme-substrate binding extend over much of the steroid skeleton.

References

Abeles, R.; Hutton, R. F.; and Westheimer, F. H. J. Am. Chem. Soc., 79:712,1957.

Abeles, R., and Westheimer, F. H. J. Am. Chem. Soc., 80:5459, 1958.

Barton, D. H. R., and Cookson, R. C. Quart. Rev., 10:44, 1956.

Burton, K., and Wilson, T. H. Biochem. J., 54:86,1953.

Englard, S. J. Biol. Chem., 233:1003,1958.

Englard, S., and Colowick, S. P. Science, 121:866, 1955.

-. J. Biol. Chem., 221:1019, 1956.

-. Ibid., 226:1047, 1957.

Eppstein, S. H.; Meister, P. D.; Murray, H. C; and Peterson, D. H.

Vitamins & Hormones, 14:359, 1956. Farrar, T. C; Gutowsky, H. S.; Alberty, R. A.; and Miller, W. G.

J. Am. Chem. Soc., 79:3978, 1957. Fisher, H. F.; Frieden, C; McKee, J. S. M.; and Alberty, R. A. J. Am.

Chem. Soc., 77:4436, 1955. Forchielli, E., and Dorfman, R. I. J. Biol. Chem., 223:443, 1956. Friedenwald, J. S., and Maengwyn-Davies, G. In: McElroy, W. D., and Glass, B. (eds.), The Mechanism of Enzyme Action, p. 180. Baltimore:

Johns Hopkins Press, 1954. Friedmann, H. C, and Vennesland, B. J. Biol. Chem., 233:1398, 1958.

Graves, J. L., and Vennesland, B. J. Biol. Chem., 226:307, 1957. Haldane, J. B. S. The Enzymes. London: Longmans, Green & Co., Ltd., 1930.

Hayabhi, O.; Sato, Y.; Jacoby, W.; and Stohlman, E. F. Arch. Biochem., 56:554,1955.

Hubener, H. J., and Lehmann, C. O. Zeitschr. physiol. Chem., 313:124, 1958.

Hurlock, B., and Talalay, P. J. Biol. Chem., 227:37, 1957.

-. Endocrinology, 62:201,1958a.

-. J. Biol. Chem., 233:886,19586.

-. Arch. Biochem. 80:468, 1959.

Jarabak, J. Unpublished observations (1959).

Kaplan, N. O. The Steric Course of Microbiological Reactions, p. 37. Ciba Foundation Study Group. London: J. & A. Churchill, Ltd., 1959.

Klvne, W. In: Klyne, W. (ed.), Progress in Stereochemistry, p. 36. London Butterworth Scientific Publications, 1954.

Koshland, D. E. In: McElroy, W. D., and Glass, B. (eds.), The Mechanism of Enzyme Action, p. 608. Baltimore: Johns Hopkins Press, 1954.

-. J. Cell. & Comp. Physiol. (Suppl. 1), 57:217, 1956.

Krasna, A. I. J. Biol. Chem., 233:1010,1958.

Lancer, L. J., and Engel, L. L. J. Biol. Chem., 233:583, 1958.

Levy, H. R.; Loewus, F. A.; and Vennesland, B. J. Am. Chem. Soc., 79:2949, 1957.

Levy, H. R., and Talalay, P. J. Am. Chem. Soc., 79:2658, 1957.

-. J. Biol. Chem., 234:2009 and 2014,1959.

Levy, H. R., and Vennesland, B. J. Biol. Chem, 228:85, 1957. Loewus, F. A.; Westheimer, F. H.; and Vennesland, B. J. Am. Chem.

Soc., 75:5018, 1953. Marcus, P. I., and Talalay, P. Proc. Roy. Soc. London, B, 144:116,1955. -. J. Biol. Chem., 205:661, 1956.

Nace, H. R., and Turner, R. B. J. Am. Chem. Soc., 75:4063, 1953. Ogston, A. G. Nature (London), 162:963,1948. Pitzer, K. S., and Beckett, C. W. J. Am. Chem. Soc., 69:977,1947. Prelog, V. The Steric Course of Microbiological Reactions, p. 79. Ciba Foundation Study Group. London: J. & A. Churchill, Ltd., 1959. Pullman, M. E.; San Peetro, A.; and Colowick, S. P. J. Biol. Chem., 206:129, 1954. Racker, E. J. Biol. Chem., 184:313,1950. San Pietro, A. J. Biol. Chem., 217:579, 1955.

Schwartz, P., and Carter, H. E. Proc. Nat. Acad. Sc. (Wash.), 40:499, 1954.

Talalay, P. Physiol. Reviews, 37:362, 1957o.

-. Record of Chem. Progress, Kresge-Hooker Library, Detroit, 18:31, 19576.

Talalay, P.; Dobson, M. M.; and Tapley, D. F. Nature (London), 170: 620,1952.

Talalay, P.; Hurlock, B.; and Williams-Ashman, H. G. Proc. Nat. Acad.

Sc. (Wash.), 44:862, 1958. Talalay, P.; Loewus, F. A.;and Vennesland, B. J. Biol. Chem., 212:801, 1955.

Talalay, P., and Marcus, P. I. J. Biol. Chem., 205:675, 1956. Talalay, P., and Williams-Ashman, H. G. Proc. Nat. Acad. Sc. (Wash.), 44:15, 1958.

Tomkins, G. M. Progress in Hormone Research, 12:125, 1956a.

-. J. Biol. Chem., 218:437,19566.

-. Ibid., 225:13, 1957.

Vennesland, B. J. Cell. & Comp. Physiol. (Suppl. 1), 47:201, 1956. -. Fed. Proc., 17:1150, 1958.

Vennesland, B., and Westheimer, F. H. In: McElroy, W. D., and Glass, B. (eds.), The Mechanism of Enzyme Action, p. 357. Baltimore: Johns Hopkins Press, 1954.

Vischer, E.; Schmtdlin, J.; and Wettstein, A. Experientia, 12:50, 1956.

Vischer, E., and Wettstein, A. Advances in Enzymology, 20:237, 1958.