On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

Abstract

The rate-limiting step in the microsomal, NADPH-dependent 24-and 26-hydroxylations of 5@-cholestane-3a,7aI,12a-triol, 60-hydroxylation of testosterone and 16n-hydroxylation of pregnenolone has been studied. Possible isotope effects were determined when the hydrogen in the substrate which is removed in the hydroxylation was substituted with deuterium; when the 4A-or 4B-hydrogen in NADPH was substituted with deuterium ; and when the incubation was carried out in deuterated water. 3. When the reactions were carried out in deuterated water, there was no significant inhibition of 24-hydroxylation of 5@-cholestane-3a,7a, 12a-tri01, whereas 16a-hydroxylation of pregnenolone was inhibited by 150/,, 6t3-hydroxylation of testosterone by 30°/, and 26-hydroxylation of 50-cholestane-3a,7a, 12a-trio1 by 60°/,.As splitting of a C-2H bond in the rate-limiting step is expected to decrease the rate of the overall reaction to less than one-half, it was concluded that the rate-limiting step in the 16a-hydroxylation of pregnenolone and 24-hydroxylation of 5/?-cholestane-3a,7a,l2a-triol is the splitting of the C-H bond in the substrate. Some other step is rate-limiting in the 6@-hydroxylation of testosterone and the 26-hydroxylation of 5,4cholestane-3a,7a, 12n-triol. I n the case of 26-hydroxylation of 5@-cholestane-3~,7a,12a-triol, water might participate in the rate-limiting step by hydration or protonolysis of the enzyme. It is suggested that a common feature of hydroxylation in which splitting of the C-H bond in the substrate is rate-limiting is a relatively low sensitivity towards carbon monoxide.The rate-limiting step in most microsomal hydroxylations has not been defined. Kinetic studies have shown that in microsomal NADPH-dependent hydroxylations of some drugs, the rate of reduction Enzymes. 3a-Hydroxysteroid dehydrogenase, fa-hydroxysteroid: NADP oxidoreductase (EC 1.1.1.50).of cytochrome P-450 -substrate complex [2-41 or the rate of oxygen activation [4] may be rate-limiting in the over-all hydroxylation reaction. I n recent work from this laboratory [5,6], it was suggested that breaking of the C-H bond in the substrate might be a rate-limiting step in the microsomal hydroxylation of some steroids and fatty acids. It was shown that 7a-hydroxylation of [7a-3H]-plus [24-14C]-labeled taurodeoxycholic acid and 9-hydroxylation of [9-2H]decanoic acid were accompanied by a kinetic isotope effect of such magnitude that it was likely that splitting of the C-3H and C 2 H bonds repectively was rate-limiting. On the other hand,

Development and Validation of a High-Throughput Radiometric Cyp3a4/5 Inhibition Assay Using Tritiated Testosterone

“…The kinetic isotope effect on T V/K was 2.3-fold. It was previously reported that 6␤-hydroxylation of deuterated testosterone proceeds in the absence of a kinetic isotope effect (Bjorkhem, 1972). This is most likely due to the difference between C-D and C-T bond energies.…”

Kinetic Deuterium Isotope Effects in Cytochrome P450 Reactions

“…KIE measurements have been made in many systems, but what new information has resulted? Many of the earliest studies were of the intermolecular competition type, and several investigators concluded that KIEs were absent or modest (Björkhem, 1972; M. Tanabe et al, 1967; Ullrich, 1969).…”

Kinetic deuterium isotope effects in cytochrome P450 oxidation reactions

“…The nature of P450 chemical reactions led to early forays into the use of KIEs as a mechanistic tool . However, many of the early studies were difficult to interpret in that (i) the studies contained mixtures of P450s and (ii) a variety of experimental designs were used . However, a high (intrinsic) D k was one of the points Groves et al .…”