Paul Talalay2 and H. G. Williams-Ashman

The Ben May Laboratory for Cancer Research and Department of Biochemistry, University of Chicago, Chicago, Illinois

I. Introduction

The over-all metabolism of steroid hormones has been the subject of many elaborate studies, especially in relation to certain clinical problems. A picture of extraordinary complexity has emerged. Relatively few primary secretory products of the gonads and adrenal cortex are converted into a vast array of metabolites which appear in the blood and are excreted in the urine. From a chemical standpoint, the principal types of metabolic changes undergone by steroid hormones in animal tissues are: oxidoreduc-tions, conjugations, and hydrolytic reactions (109). The oxidations and reductions may be further classified into four main categories: (a) inter-conversions of hydroxyl and ketone functions, (b) introduction or hy-drogenation of carbon-to-carbon double bonds, (c) hydroxylations, and (d) oxidative scission of side chains. Both alcoholic and phenolic hydroxyl groups may become conjugated with sulfate or glucuronide residues. The resulting conjugates are hydrolyzed by various sulfatases and glucuronidases.

Apart from their role in the biosynthesis of hormones by the adrenals and gonads, oxidoreductions of steroid hormones occur in the liver and many peripheral tissues (11, 12, 107). Conjugations to form water-soluble sulfates and glucuronosides appear to take place principally in hepatic cells. The fact that the hormonal activity of most of these metabolites is grossly inferior to that of the parent hormones lies at the basis of the concept that the liver and peripheral tissues "inactivate" steroid hormones.

What is the physiological significance of these reactions? Are they all primarily homeostatic devices to rid the organism of excessive amounts of hormones, and to promote their excretion? Do any of them facilitate the transformation of steroids into forms with greater or perhaps more specialized hormonal activity, or their transport to proper intracellular locations? May some of them be a direct consequence of the mechanisms by which these internal secretions modify the growth and function of cells? The answers to these important questions require a proper understanding of the biochemistry of these transformations. Studies of the metabolic changes of steroid hormones carried out by whole animals (28), perfused organs (43) and cells grown in culture (107) cannot, by their very nature, provide much information about the intricacies of the enzymatic machinery involved. Until recently, most studies of steroid transformations by slices or cell-free extracts of animal tissues in vitro have dealt largely with the chemical nature of the products (28).

1 Original investigations from the authors' laboratories were supported by the American Cancer Society.

2 Supported by a permanent faculty-level grant from the American Cancer Society.

Our knowledge of the enzymatic mechanisms in steroid metabolism is most fragmentary (109, 110). The hydroxylation of steroids at some positions in the molecule can be demonstrated with cell-free preparations, and, in most instances, TPNH3 and molecular oxygen are required (86). But none of the enzymes concerned have been purified extensively, and both the chemical nature of the intermediates and the mode of activation of molecular oxygen are obscure. Enzymes which catalyze the introduction or hydrogen-ation of carbon-to-carbon double bonds in the steroid nucleus have been isolated from a number of sources in a relatively crude state; their specificity for hydrogen donors and acceptors has been examined, but relatively little is known about their mechanism of action (77, 121). The constitution of the nucleotide structures which act as glucuronyl and sulfuryl donors has been determined, and some of the transferases catalyzing the union of these groups with steroids have been partially purified (58, 95, 99). An enzyme which catalyzes the isomerization of the double bond of β,y-unsaturated 3-ketosteroids and causes an intramolecular hydrogen shift (116) has been crystallized from bacteria (68). The hydroxysteroid dehydrogenases catalyze the reversible oxidation of alcoholic groups to carbonyl functions with pyridine nucleotides as hydrogen acceptors, and a few of these enzymes have been isolated in a highly purified state (83, 110, 115). The equilibria established by some of these enzymes are known with certainty, and there is precise information about the binding constants for both nucleotides and steroids. Of related interest are measurements of the complexing of steroids with well-characterized proteins (135), and also with purines and their nucleotides (91).

Our recent finding that catalytic amounts of some steroid hormones mediate hydrogen transfer between DPN and TPN in the presence of certain mammalian hydroxysteroid dehydrogenases (55, 112, 117) suggested that this coenzyme-like property of the hormones may be related to their regulatory function. Whether there is any heuristic value to this suggestion can be decided only after much further experimentation. In this paper we will consider the chemistry of transhydrogenations catalyzed by hydroxysteroid dehydrogenases, knowledge of which is an absolute prerequisite to any investigation of their wider physiological significance. Model reactions with similar enzymes of bacterial origin have cast considerable light on the mechanism of action of steroid-dependent transhydrogenating systems of animal tissues. We will also consider model reactions catalyzed by certain oxidizing enzymes in which catalytic levels of phenolic estrogens (both natural and synthetic) mediate hydrogen transport between molecules of physiological interest.

3 The following abbreviations are used: DPN, DPNH and TPN, TPNH for the oxidized and reduced forms of di- and triphosphopyridine nucleotides, respectively; Tris for tris(hydroxymethyl) aminomethane.

II. Hydroxysteroid Dehydrogenases

Metabolic interconversions of hydroxy- and ketosteroids occur widely in nature, and have been described in a variety of animal tissues and in microorganisms. The term hydroxysteroid dehydrogenases was proposed for those pyridine nucleotide-linked enzymes which catalyze reversible oxidations of hydroxyl functions on the steroid skeleton and side chain (109). These catalysts exhibit a high degree of specificity with respect to the position and steric configuration of the hydroxyl group undergoing oxidation. The reactions may be formulated as follows: steroid alcohol + DPN+ (TPN + ) ⇋ steroid ketone + DPNH (TPNH)+ H+

Several hydroxysteroid dehydrogenases have been purified in varying degree and characterized with respect to their intracellular localization and substrate and pyridine nucleotide specificity. The soluble, adaptive 3a-hydroxysteroid dehydrogenase of Pseudomonas testosteroni, grown on testosterone, is one of the most highly purified and well-characterized enzymes of this class (activity = 50 µmoles DPN reduced per minute per milligram protein at 25°C.) (83). This enzyme reacts with DPN or some of its analogs but is inert toward TPN. It oxidizes 3a-hydroxyl groups of C19, C21, and C24 steroids, in which the A:B ring fusion may be cis or trans. The Michaelis constant for androsterone is 1.5 X 10-6M. Steroids in which the A:B ring fusion is cis are more slowly oxidized and less firmly bound to the enzyme. A DPN-linked 3a-hydroxysteroid dehydrogenase from Escherichia jreundii reacts preferentially with bile acids and is inert toward C19 steroids (42). Similar enzymes have been found in the livers of various species. A soluble 3a-hydroxysteroid dehydrogenase has been purified from rat liver by Tomkins (120). This enzyme reacts equally well with DPN and TPN and attacks 3a-hydroxyl groups of C19 and C21 steroids, although in contrast to the Pseudotnonas enzyme, the A:B cis steroids react more rapidly than the A:B trans compounds (55, 120). Liver microsomes also contain a firmly bound 3a-hydroxysteroid dehydrogenase which reacts with both pyridine nucleotides (56).