PubMed · 3724073
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M A Edwards, W Giguiere, D Lewis, R C Baselt. Intoxilyzer interference by solvents.. https://doi.org/10.1093/jat%2F10.3.125
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6-Methylsalicylic acid synthase, the multifunctional enzyme complex that catalyzes the biosynthesis of the tetraketide 6-methylsalicylic acid, was modified by thiol-specific inhibitors and cross-linking reagents. Treatment with 1,3-dibromopropan-2-one caused rapid enzyme inactivation and formation of cross-linked dimers. Analysis by SDS-PAGE, density gradient ultracentrifugation, and secondary modification with [14C]iodoacetamide showed that two types of cross-linked dimers were formed. Peptides derived from native and 1,3-dibromopropan[2-14C]one-treated enzyme were isolated by SDS-PAGE and N-terminally sequenced. The sequences of the two N-termini from cross-linked peptides were located in the nucleotide-derived amino acid sequence and found to arise from the beta-ketoacyl synthase and acyl carrier protein components of the 6-methylsalicylic acid synthase subunit. Acetyl-CoA protected the enzyme from both inactivation and cross-linking by binding to the reactive cysteine of the beta-ketoacyl synthase component. Malonyl-CoA protected against cross-linking by binding to the thiol moiety of the 4'-phosphopantetheine prosthetic group of the acyl carrier protein. Formation of a mixed disulfide on treatment with 5,5'-dithiobis(2-nitrobenzoic acid) indicated that these two types of thiol residue are positioned close to each other in the active enzyme. From these studies, it was concluded that two pairs of functional dimers are present in the 6-methylsalicylic acid synthase tetramer and that, within each dimer, the beta-ketoacyl synthase and acyl carrier protein components are juxtaposed to allow the respective cysteine (residue 204) and 4'-phosphopantetheine thiols to interact during condensation. This spatial arrangement of thiols at the condensing active site is analogous to that found in type I vertebrate fatty acid synthases and other polyketide synthases.
A variety of trifluoromethyl ketones (TFMKs) have been studied as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis. The chemicals behaved as tight slow-binding inhibitors, the beta-thio derivatives being the most potent ones, particularly 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) with an IC50 of 0.08 microM. Other TFMKs, containing a propenyl group at the non-polar end of the molecule as in the natural pheromone structure (compounds 4, 8 and 9), were also notably active. Correlation studies of inhibition with lipophilicity (log P) indicated that the optimum log P values for activity of the beta-thio compounds are in the range of 3.03-5.11, while a higher lipophilicity (range 5.37-5.89) was required among the devoid of sulfur ketones. 19F NMR studies, carried out with OTFP, showed that the inhibitor binds the enzyme in a reversible manner, forming an adduct (probably a hemiacetal) of tetrahedral geometry with the active site of the enzyme. To our knowledge, this is the first time that such a study on a natural esterase extract has been undertaken.
Human, monkey, and rat liver microsomes catalyze bisallylic hydroxylations of arachidonic and linoleic acids. The cytochrome P450 gene family of these hydroxylases has not been determined. We examined whether inducers of cytochrome P450 could augment the bisallylic hydroxylation activity of male rat liver microsomes. The microsomes were incubated with [14C]linoleic acid and NADPH and the monohydroxy metabolites were characterized. Microsomes prepared from control rats yielded mainly 18-hydroxyoctadecadienoic acid (18-HODE) and 17-HODE and microsomes from clofibrate-treated rats 18-HODE. Microsomes from beta-naphthoflavone-treated rats hydroxylated linoleic acid without position specificity, i.e., at carbons 8, 11, 14, 16, 17, and 18. 11-HODE, 17-HODE, and 18-HODE were major metabolites. Microsomes from rats treated with phenobarbital, isopropanol, imidazole, or acetone also formed these three products along with many other hydroxy metabolites. The synthetic glucocorticoid dexamethasone increased the biosynthesis of 11-HODE selectively. Microsomes from male Sprague-Dawley and Fischer rats treated with dexamethasone mainly formed 11-HODE and 18-HODE. The biosynthesis of 11-HODE was increased 10-fold and troleandomycin (50 microM) inhibited the biosynthesis of 11-HODE by 90%. The bisallylic hydroxylases were also investigated with 14C-labeled arachidonic and eicosapentaenoic acids as substrates. Microsomes from rats treated with dexamethasone converted 20:4n-6 to 13-hydroxyeicosatetraenoic acid (13-HETE), 10-HETE, 7-HETE, 19-HETE, and 20-HETE. Induction by acetone yielded the same products. Microsomes from dexamethasone-treated rats metabolized 20:5n-3 to 16-hydroxyeicosapentaenoic acid (16-HEPE), 13-HEPE, 10-HEPE, 19-HEPE, and 20-HEPE as major products, while microsomes from control and acetone-treated rats mainly formed 19-HEPE and 20-HEPE. We conclude that microsomes from dexamethasone-treated rats catalyze bisallylic hydroxylations of 18:2n-6, 20:4n-6, and 20:5n-3, possibly by induction of bisallylic hydroxylases of the CYP3A subfamily.