Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

Abstract

Testosterone 6␤-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6␤-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6␤-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-2 H 5 ; (ii) 6,6-2 H 2 ; (iii) 6␣-2 H; (iv) 6␤-2 H; and (v) 6␤-3 H testosterone. Only the 6␤-hydrogen was removed by P450 3A4 and not the 6␣, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the ␤ face. Analysis of the rates of hydroxylation of 6␤-1 H-, 6␤-2 H-, and 6␤-3 Hlabeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ( D k) of 15. The deuterium isotope effects on k cat and k cat /K m in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6␤-2 H substitution. The high D k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high D k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high D k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking. P4501 enzymes are found in organisms from Archebacter to humans and catalyze oxidations of a great variety of chemicals (1, 2). The human P450s are the major enzymes involved in the clearance of drugs, and P450 3A4, the most abundant P450 in liver and small intestine, is involved in the oxidation of one-half of the drugs used today (3, 4). Two crystal structures of P450 3A4 have been published recently (5, 6); however, neither has a substrate bound in a position amenable to oxidation. P450 3A4 exhibits heterotropic and homotropic cooperativity in some but not all of its reactions (4, 7-10), and several models have been proposed to explain such behavior (9,(11)(12)(13)(14)(15). A view frequently expressed for smaller substrates is that binding is relatively loose and the regioselectivity of oxidation of substrates is largely a function of the ease of C-H bond breaking (16,17).Relatively little information is available regarding what step is rate-limiting in P450 3A4 reactions. The addition of the first electron in the catalytic cycle (step 2 of Scheme 1) is apparently relatively fast in the presence of an optimal concentration of NADPH-P450 reductase (18). Only limited information has been obtained regarding steps 1 and 3-9 (Scheme 1) with P450 3A4 reactions. Without information on the extent to which chemical step...

Mechanisms of cytochrome P450 substrate oxidation: MiniReview

“…An important finding in this work is the slower events in substrate binding are in the same timescale as other steps (e.g., reduction [29]) and even the overall substration oxidation process [29,35]. It is proposed that this "twisting and turning" of P450 3A4 while hydroxylation proceeds is an important part of the anomalous cooperative behavior of P450 3A4 [29].…”

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

“…However, it is also active in the oxidations of many steroids, including cholesterol (13), estradiol (14), progesterone (15), cortisol (16), and testosterone and androstenedione (14,15). The major reaction observed with testosterone is 6␤-hydroxylation (14,15,17); hydroxylation at the 2␤, 1␤, and 15␤ sites also occurs (17,18). However, no physiological relevance of any of these hydroxylations has been established.…”

A malleable catalyst dominates the metabolism of drugs

“…First, the availability of multiple modes of loose binding (e.g., in the erythromycin structure) can explain some of the ''loose'' regioselectivity of P450 3A4. However, the view that the active site cavity is completely noninstructive must be rejected: the ligand ketoconazole shows interactions with amino acid residues in P450 3A4 (1); work from our own laboratory shows the striking stereoselectivity of ␤ vs. ␣ hydrogen abstraction in testosterone 6␤-hydroxylation (15). Nevertheless, the availability of alternate and nonproductive binding modes can frustrate various biophysical approaches, in which stoichiometry measurements may or may not reflect functional interactions (8,16).…”