Cytochrome P-450scc-mediated oxidation of (20S)-22-thiacholesterol: Characterization of mechanism-based inhibition

Abstract

(20S)-22-thiacholesterol (1) is found to be a potent competitive inhibitor of pregnenolone biosynthesis from cholesterol by purified reconstituted bovine adrenal cytochrome P-450scc. The apparent dissociation constant Kd, determined from difference spectra, is 0.6 microM, close to the value from kinetic studies for the apparent inhibition constant, Ki, of 0.8 microM. Studies of the time course of pregnenolone production indicate that under turnover conditions the competitive inhibitor (1) is converted to a tighter binding inhibitor, shown to be (20S,22R)-22-thiacholesterol S-oxide (4), with high diastereoselectivity and in a time-dependent manner. Both the diastereomeric sulfoxides, (20S,22S)-22-thiacholesterol S-oxide (3) and (20S,22R)-22-thiacholesterol S-oxide (4), exhibit properties consistent with their being competitive versus cholesterol, but the (22R)-sulfoxide (4) binds approximately 10 times more tightly than the (22S) diastereomer (3). The apparent Kd values of sulfoxides 4 and 3 are 0.1 and 1.14 microM, respectively. EPR and absorption spectroscopic studies of enzyme-inhibitor complexes suggest direct coordination of the oxygen atom of the (22R)-sulfoxide (4) with the catalytic heme center. This implies that the inhibitor operates by directly blocking further reaction at the active site heme group, with a substantial lifetime of the enzyme-inhibitor complex.

Pentasilatricyclo[2.1.0.02,5]pentane and Its Anion

“…In conclusion, care must be exercized in utilizing thioethers to probe the stereochemical outcomes of more energetically demanding reactions catalyzed by P450s, such as CH oxidation. The two prior reports of comparable P450‐mediated sulfur or methylene oxidation suggest that they can proceed with comparable stereochemical outcomes,17, 18, 21 but our results demonstrate that this is not universal. The postulate that Cpd 0 is responsible for thioether oxidation in P450s is in harmony with the work of Jones and co‐workers that suggests that two different oxidants function in a P450 BM3 ‐catalyzed oxidation of an amine or thioether 8.…”

Is the Ferric Hydroperoxy Species Responsible for Sulfur Oxidation in Cytochrome P450s?

“…The two prior reports of comparable P450-mediated sulfur or methylene oxidation suggest that they can proceed with comparable stereochemical outcomes, [17,18,21] but our results demonstrate that this is not universal. The postulate that Cpd 0 is responsible for thioether oxidation in P450s is in harmony with the work of Jones and co-workers that suggests that two different oxidants function in a P450 BM3 -catalyzed oxidation of an amine or thioether.…”

“…This drop in selectivity may be expected as the more easily oxidized sulfur atom may allow reaction from a greater range of reactive conformations than the more demanding C À H bond. [17][18][19] Intriguingly, however, the sulfoxides were produced with the opposite enantioselectivity to that observed for comparable fatty acid hydroxylation (S sulfoxides versus R alcohols). [21,22] One explanation for this switch in enantiomeric preference for oxidation 3 and 4 relative to the analogous fatty acids is that they were accessing an unusual binding conformation that allowed the production of the S sulfoxide.…”

“…[11,13,16] Two isolated examples of P450-catalyzed oxidation of analogous thioetheror methylene-containing substrates appeared to indicate that these reactions proceed with the same stereochemical outcome. [17][18][19] However, we felt it prudent to validate the use of thiafatty acids as stereochemical probes with fatty acid metabolizing P450s for which the stereochemical outcomes were known. Thus, we chose to investigate thiafatty acid oxidation by the widely studied P450 BM3 , a catalytically selfsufficient P450 isolated from Bacillus megaterium.…”

The biosynthesis of steroids and triterpenoids

“…527,528 (20S)-22-Thiacholesterol [although not the (20R) diastereoisomer] is a mechanism-based inhibitor of this enzyme. 529 A steroidal mechanism-based fluorogenic probe has been synthesised and used to confirm the location of P-450 scc on the inner surface of mitochondrial membranes. 530 The interactions between this membrane-bound enzyme and ferredoxin, which mediates electron transfer to the enzyme, have been shown to be electrostatic in nature 531 and to depend on the presence of specific charged amino acids.…”