David L. Corina

The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14α-Demethylase of Candida albicans (Other Names Are: Lanosterol 14α-Demethylase, P-45014DM, and CYP51)

The Candida albicans sterol 14α-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 μmol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3β-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17α-hydroxylase-17,20-lyase (P-450,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered.

Incorporation of label from 18O2 into acetate during side-chain cleavage catalysed by cytochrome P-45017α(17α-hydroxylase-17,20-lyase)

Samples of pregnenolone and 17α-hydroxypregnenolone deuteriated at the C-21 methyl group have been prepared and subjected to side-chain cleavage with a pig testes microsomal preparation under 18O2. 17α-Hydroxypregnenolone was exclusively cleaved to dehydroisoandrosterone and the acetic acid released during the process was found to incorporate 0.90 atom of 18O. When a similar incubation was performed with pregnenolone two steroidal products, dehydroisoandrosterone and androsta-5,16-dien-3β-ol, were formed in an approximate ratio of 1:2–3 and the acetic acid formed in the process was again shown to contain 0.85–0.90 atom of 18O. Complementary experiments in which the two substrates labelled with 18O at the C-20 carbonyl group were incubated under 16O2 gave acetic acid retaining between 65–85% of the original carbonyl oxygen. The experiments strengthen the hypothesis that the two C(17)–C(20) bond cleavages described above correspond to the acyl-carbon fission process of the equation below showing the indicated fate of the various oxygen atoms: [graphic omitted]

The phospholipid annulus of mitochondrial NADH—ubiquinone reductase A dual phospholipid requirement for enzyme activity

A phospholipid requirement for the activity of several membrane-bound enzymes has been clearly established, e.g. [1,2] but the evidence for a phospholipid dependence of mitochondrial NADH dehydrogenase is conflicting at present. Reversible loss of NADH-ubiquinone-1 oxidoreductase activity following removal of phospholipids by phospholipase A treatment [3,4] or cholate and ammonium sulphate extraction [5] has been reported, but a water-soluble, lipid-free NADH dehydrogenase catalyzing ubiquinone reduction has been isolated by Baugh and King [6]. An approach to the study of lipid-protein interactions which avoids the potential problem of irreversible denaturation on removal of lipids is the lipid-substitution technique of Warren et al. [7]. We have applied this method to purified NADH-ubiquinone oxidoreductase (Complex I) [8] which is isolated as a lipoprotein complex. We are interested in investigating the role of lipids in mitochondrial electron transport for reasons other than the conflicting views on NADH dehydrogenase. Firstly, the respiratory chain complexes contain relatively high levels of cardiolipin whose specialized function (if any) is unknown. Secondly, the means by which respiratory complexes interact in the membrane is unclear. For example although ubiquinone-lO behaves as a kinetically homogeneous pool mediating hydrogen transfer from (separate) Complex I to Complex III [9], this picture is difficult to reconcile with the formation of a 1 : 1 binary complex from

Mechanistic studies on pregnene side-chain cleavage enzyme (17α-hydroxylase-17,20-lyase) using 18O

In the conversion of pregnenolone 1 into dehydroisoandrosterone 3 and 3β-hydroxyandrosta-5,16-diene 5 an atom of oxygen from O2 is incorporated into the side chain released as acetate; during the conversions the C-21 methyl hydrogens of the precursor are not disturbed, the status of 16α and 17α hydrogen atoms during the conversions is also defined.

The mechanism of cytochrome P-450 dependent C–C bond cleavage: studies on 17α-hydroxylase-17,20-lyase

It is shown that formation of 17α-hydroxyandrost-5-en-3β-ol from pregnenolone occurs solely at the expense of the cleavage of the C-17–C-20 bond of the precursor and is best rationalised by invoking the participation of an FeIII–OOH intermediate in the acyl–carbon fission process.

Gas chromatographic and gas chromatographic—mass spectrometric characterisation of some thiosulphonates and polymethylene dimethane thiosulphonates

Abstract The characterisation by gas chromatography (GC) and GC—mass spectrometry (MS) of thiosulphonates of the types R1·SO2S·R2 (type I) and CH3·SO2S·(CH2)n S·SO2CH3 (type II) is described. Type I thiosulphonates showed suitable GC—MS properties as the intact molecules. Type II could be characterised by GC—MS only after reduction to the dithiol; direct probe spectra were necessary to characterise all the intact type II compounds by MS.

Mechanism of C-2–C-3 bond formation and cleavage in serine transhydroxymethylase reactions

It is shown that in the serine transhydroxy-methylase-catalysed aldol cleavage of threonine containing 18O at the hydroxy-group the label is quantitatively transferred to acetaldehyde.

The status of oxygen atoms in the removal of C-19 in oestrogen biosynthesis

Three O2-dependent reactions are involved in the removal of C-19 as formate, in oestrogen biosynthesis; it is shown that the oxygen atoms introduced in steps 1 and 3 of the proces are the ones which are found in the biosynthetic formate.

Gas chromatographic and mass spectrometric properties of naphthalenemethyl esters and cyclohexanemethyl esters of organic acids

Abstract The preparation of the esterifying reagents naphthyldiazomethane and cyclohexyldiazomethane is described. These reagents have been used to prepare the naphthalenemethyl and cyclohexanemethyl esters of various types of carboxylic acids. Data are presented for the gas chromatographic retention times of the esters on OV-1 and also for their mass spectral fragmentation patterns.

3-Hydroxy-3-methylglutaric aciduria: a possible pitfall in diagnosis.

The mitochondrial enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) is a component of the pathway for leucine catabolism and, in the liver, has a key role in the synthesis of the ketone body, acetoacetate [l]. Babies with an inherited deficiency of this enzyme (McKusick 24645) generally present during the first year of life with episodic severe hypoglycaemia and metabolic acidosis, but no ketosis, which may progress to coma and death [l-4]. Fatty infiltration of the liver has been reported in the acute illness [4,5]. With prompt diagnosis and dietary management, and avoidance of prolonged fasting, the prognosis may be good. There is a characteristic organic aciduria, with excretion of large amounts of 3-hydroxy-3-methylglutaric (HMG), 3-methylglutaconic, 3-methylglutaric and 3-hydroxyisovaleric acids and sometimes, during acute epidoses, increases in hexanedioic and glutaric acids and excretion of 3-methylcrotonylglycine [1,3,6]. Seriously ill babies with an acquired deficiency of the enzyme have also been reported [7-91. We recently investigated a 5-month-old baby, who had a cot death, for a possible inherited metabolic disorder. His liver was grossly enlarged and fatty. Initial examination of a urinary extract for organic acids by gas chromatography suggested the possibility of HMG-CoA lyase deficiency, but the HMG peak was small in relation to 3-methylglutaconic acid. Fourteen days later, analysis of the same urine extract