The conversion of cholest-7-en-3beta-ol into cholesterol. General comments on the mechanism of the introduction of double bonds in enzymic reactions

Convenient syntheses of 6β-tritiated Δ⁷-cholestenol and 3α-tritiated Δ⁷-cholestene-3β,5α-diol are described. It was shown that the conversion of 6β-tritiated Δ⁷-cholestenol into cholesterol is accompanied by the complete retention of label. It was unambiguously established that the overall reaction leading to the introduction of the double bond in the 5,6-position in cholesterol occurs via a cis-elimination involving the 5α- and 6α-hydrogen atoms and that during this process the 6β-hydrogen atom remains completely undisturbed. Metabolic studies with 3α-tritiated Δ⁷-cholestene-3β,5α-diol revealed that under anaerobic conditions the compound is not converted into cholesterol. This observation, coupled with the previous work of Slaytor & Bloch (1965), is interpreted to exclude a hydroxylation–dehydration mechanism for the origin of the 5,6-double bond in cholesterol. It was also shown that under aerobic conditions 3α-tritiated Δ⁷-cholestene-3β,5α-diol is efficiently converted into cholesterol and that this conversion occurs through the intermediacy of 7-dehydrocholesterol. Cumulative experimental evidence presented in this paper and elsewhere is used to suggest that the 5,6-double bond in cholesterol originates through an oxygen-dependent dehydrogenation process and a hypothetical mechanism for this and related reactions is outlined.     

The stereochemistry of the hydrogen elimination in the biological conversion of cholest-7-en-3β-ol into cholesterol

1. The syntheses of Δ⁷-[4-¹⁴C]cholestenol (XVI, Scheme 3) and Δ⁷-[6α-³H]-cholestenol (XII, Scheme 2) are described. 2. The metabolism of doubly labelled Δ⁷-cholestenol (II, Scheme 1) by rat-liver homogenates was studied. 3. During the enzymic conversion of Δ⁷-cholestenol into cholesterol (IV, Scheme 1) the 6α-hydrogen atom of the former is lost and the overall reaction corresponds to a cis-elimination. 4. In the light of these results various mechanisms for the conversion of Δ⁷-cholestenol into cholesterol are discussed.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

The impact of aromatase mechanism on other P450s

Experimental findings from a number of laboratories have converged to show that the conversion of androgens into oestrogen, catalysed by aromatase, involves three distinct reactions which occur at a single active site. That each one of these reactions belongs to a different generic type was revealed by chemical consideration, together with our ¹⁸O-experiments. In particular, these findings high-lighted the fact that the third reaction in the sequence occurs by a novel process for which a number of plausible mechanisms have been considered. The scrutiny of these mechanisms has involved either studies on aromatase itself, or on related enzymes which catalyse the aromatase type of cleavage reaction as generalized in equation 1:

Full-size image

The acyl-carbon cleavage reaction of equation 1 is catalysed by sterol 14-demethylases, accounts for several side-chain fission products formed by CYP17 (17-hydroxylase-17,20-lyase), and constitutes a weak property of certain drug metabolizing P450s, when given aliphatic aldehydes as substrates. From cumulative studies on these enzymes, consensus is beginning to emerge that the acyl-carbon fission may be promoted by the Fe^III---OOH intermediate, formed during the catalytic cycles of P450s. The precedent for the direct involvement of the Fe^III---OOH species in the reaction of equation 1 is influencing our thinking regarding the mechanism of the conventional hydroxylation reaction. The status of knowledge surrounding the current debate on these issues will be reviewed.     

Requirement for calcium ions in acetylcholine-stimulated phosphodiesteratic cleavage of phosphatidyl-myo-inositol 4,5-bisphosphate in rabbit iris smooth muscle

1. The mechanism of acetylcholine-stimulated breakdown of phosphatidyl-myo-inositol 4,5-bisphosphate and its dependence on extracellular Ca(2+) was investigated in the rabbit iris smooth muscle. 2. Acetylcholine (50mum) increased the breakdown of phosphatidylinositol bisphosphate in [(3)H]inositol-labelled muscle by 28% and the labelling of phosphatidylinositol by 24% of that of the control. Under the same experimental conditions there was a 33 and 48% increase in the production of (3)H-labelled inositol trisphosphate and inositol monophosphate respectively. Similarly carbamoylcholine and ionophore A23187 increased the production of these water-soluble inositol phosphates. Little change was observed in the (3)H radioactivity of inositol bisphosphate. 3. Both inositol trisphosphatase and inositol monophosphatase were demonstrated in subcellular fractions of this tissue and the specific activity of the former was severalfold higher than that of the latter. 4. The acetylcholine-stimulated production of inositol trisphosphate and inositol monophosphate was inhibited by atropine (20mum), but not tubocurarine (100mum); and it was abolished by depletion of extracellular Ca(2+) with EGTA, but restored on addition of low concentrations of Ca(2+) (20mum). 5. Calcium-antagonistic agents, such as verapamil (20mum), dibenamine (20mum) or La(3+) (2mm), also abolished the production of the water-soluble inositol phosphates in response to acetylcholine. 6. Release of inositol trisphosphate from exogenous phosphatidylinositol bisphosphate by iris muscle microsomal fraction (;microsomes') was stimulated by 43% in the presence of 50mum-Ca(2+). 7. The results indicate that increased Ca(2+) influx into the iris smooth muscle by acetylcholine and ionophore A23187 markedly activates phosphatidylinositol bisphosphate phosphodiesterase and subsequently increases the production of inositol trisphosphate and its hydrolytic product inositol monophosphate. The marked increase observed in the production of inositol monophosphate could also result from Ca(2+) activation of phosphatidylinositol phosphodiesterase. However, there was no concomitant decrease in the (3)H radioactivity of this phospholipid.     

Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein.

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].     

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanistic studies on C-19 demethylation in oestrogen biosynthesis

Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O₂ and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with ²H in addition to ¹⁸O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[¹⁸O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent ¹⁶O substrates were aromatized under ¹⁸O₂, the formic acid from both substrates contained one atom of ¹⁸O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O₂ is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10β-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10–C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.     

Dual infections of Culex tritaeniorhynchus with West Nile virus and Nosema algerae

Culex tritaeniorhynchus females infected with the microsporidian Nosema algerae, and uninfected control females were compared for susceptibility to infection with West Nile (WN) virus and for the ability to transmit virus. When fed on a high titered dose fo virus, 95% of the control females became infected, whereas only 65% of the N. algerae-infected females were infected with WN virus. However, at two lower viral doses, no differences in susceptibility were observed. No significant differences in transmission ability were found between the N. algerae-infected and control females when tested at 10, 14, and 21 days after infection with WN virus. Also, in mosquitoes dually infected with N. algerae and WN virus, neither agent affected the ability of the other to replicate.