The mechanism of inactivation of lipoxygenases by acetylenic fatty acids

The inactivation of soybean lipoxygenase by 5,8,11,14-eicosatetraynoic acid was studied in detail. The inactivation was found to be time-dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme-product complex, yielded a Km value for 5,8,11,14-eicosatetraynoic acid of 1.3 microM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s-1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14-eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14-eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14-eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14-eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13LS-hydroperoxy-linoleic acid. These results, as well as the lack of covalent binding of 14C-labelled 5,8,11,14-eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D.T. Downing, D.G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218-223; K.H. Gibson (1977) Chem. Soc. Rev. 6, 489-510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.     

Mechanism-based in vivo inactivation of lauric acid hydroxylases

The hepatic cytochrome P-450 isozymes that catalyze omega- and (omega - 1)-hydroxylation of lauric acid are specifically inactivated in vitro but not in vivo by 10-undecynoic acid. The lack of in vivo activity may result from rapid degradation of the inhibitor by beta-oxidation. Strategies for the construction of fatty acid analogues that retain the ability to inactivate fatty acid hydroxylases but are resistant to metabolic degradation have therefore been sought. Fatty acid analogues in which the carboxylic acid group is replaced by a sulfate moiety, or in which two methyl groups are placed vicinal to the carboxylic acid group, have been found to inactivate lauric acid hydroxylases in vitro and in vivo without causing time-dependent inhibition of ethoxycoumarin O-deethylation or N-methyl-p-chloroaniline N-demethylation.     

Microsomal glycerolphosphate acyltransferase inactivation by fatty acids

Glycerolphosphate acyltransferase activity in microsomes from rat adipose tissue is shown to decrease with time upon incubation with adipose tissue cytosolic fraction. The inactivation can be prevented with serum albumin and seems to be caused by an increase in endogenous free fatty acid as a consequence of the action of cytosolic lipase(s) on the membrane lipids. Similar inactivation can be observed after short incubation of microsomes with oleic acid at micromolar concentrations. Diacylglycerol acyltransferase is also inhibited by oleic acid, although to a lesser degree. In contrast, glucose-6-phosphatase and NADPH-cytochrome reductase activities are not changed. The oleic acid effect appears to occur upon binding to the microsomal membranes and can be prevented by bovine serum albumin at protein/fatty acid molar ratios above one. These results suggest that free fatty acids may be involved in the modulation of triacylglycerol synthetic enzymes.     

Differential inactivation of plant lauric acid omega- and in-chain-hydroxylases by terminally unsaturated fatty acids

The microsomal fraction from Vicia sativa L. cv. Septimane contains a cytochrome P-450-dependent lauric acid omega-hydroxylase that is inactivated in a time-dependent, pseudo-first-order manner when the microsomes are incubated with 11-dodecynoic acid. The rate constant for the inactivation is approximately 4.3-4.8 X 10(-3) s-1. In contrast, the olefinic analog 11-dodecenoic acid is primarily a time-independent inhibitor of the omega-hydroxylase. 1-Aminobenzotriazole, 3-phenoxy-1-propyne, and 3-(2,4-dichlorophenoxy)-1-propyne, mechanism-based inactivators of cinnamic acid 4-hydroxylase, and 9-decenoic acid, a mechanism-based inactivator of the lauric acid in-chain hydroxylase, are at best poor inactivators of the omega-hydroxylase. Conversely, cinnamic acid 4-hydroxylase is only slightly affected by concentrations of 11-dodecynoic acid that completely inactivate the omega-hydroxylase. 11-Dodecynoic acid is thus a potent, relatively specific, inactivator of the V. sativa lauric acid omega-hydroxylase.     

The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids

Cytochrome P-450LA omega purified from clofibrate-induced rat liver oxidizes lauric acid to 11- and 12-hydroxydodecanoic acid in approximately a 1:17 ratio at a rate of 20 nmol/nmol P-450/min. In contrast, cytochrome P-450b oxidizes lauric acid much more slowly (0.5 nmol/nmol P-450/min) to an 8:1 mixture of the same metabolites. Western blot analysis indicates that P-450LA omega accounts for 1-2 and 16-30%, respectively, of the total cytochrome P-450 in uninduced and clofibrate-induced rat liver. Cytochrome b5 increases the efficiency of omega-hydroxylation but not the rate of catalytic turnover. Incubation of the enzyme with 10-undecynoic acid (10-UDYA) results in loss of approximately 45% of the enzymatic activity but none of the enzyme chromophore. Approximately 1 mol of 1,11-undecandioic acid is produced per mole of inactivated enzyme. This extraordinary inactivation efficiency is confirmed by NADPH consumption studies. Approximately 0.5 equivalents of label are covalently bound to the enzyme when it is incubated with 14C-labeled 10-UDYA. 11-Dodecenoic acid appears not to be a substrate for cytochrome P-450LA omega but is oxidized, presumably by a contaminating isozyme, to a 10:1 mixture of 11,12-epoxydodecanoic acid and 12-oxododecanoic acid. The results suggest the presence of two closely related P-450LA omega enzymes, only one of which is susceptible to inactivation by 10-UDYA. They also indicate that cytochrome P-450LA omega has a highly structured active site that sterically suppresses omega-1-hydroxylation in order to deliver the oxygen to the thermodynamically disfavored terminal carbon. Protein rather than heme alkylation follows from this reaction regiospecificity.     

Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana

Towards defining the function of Arabidopsis thaliana fatty acid hydroxylases, five members of the CYP86A subfamily have been heterologously expressed in baculovirus-infected Sf9 cells and tested for their ability to bind a range of fatty acids including unsubstituted (lauric acid (C12:0) and oleic acid (C18:1)) and oxygenated (9,10-epoxystearic acid and 9,10-dihydroxystearic acid). Comparison between these five P450s at constant P450 content over a range of concentrations for individual fatty acids indicates that binding of different fatty acids to CYP86A2 always results in a higher proportion of high spin state heme than binding titrations conducted with CYP86A1 or CYP86A4. In comparison to these three, CYP86A7 and CYP86A8 produce extremely low proportions of high spin state heme even with the most effectively bound fatty acids. In addition to their previously demonstrated lauric acid hydroxylase activities, all CYP86A proteins are capable of hydroxylating oleic acid but not oxygenated 9,10-epoxystearic acid. Homology models have been built for these five enzymes that metabolize unsubstituted fatty acids and sometimes bind oxygenated fatty acids. Comparison of the substrate binding modes and predicted substrate access channels indicate that all use channel pw2a consistent with the crystal structures and models of other fatty acid-metabolizing P450s in bacteria and mammals. Among these P450s, those that bind internally oxygenated fatty acids contain polar residues in their substrate binding cavity that help stabilize these charged/polar groups within their largely hydrophobic catalytic site.     

Cytochrome P450-dependent fatty acid hydroxylases in plants

In plants, hydroxy-fatty acid production is mainly the result of enzymatic reactions catalyzed by cytochrome P450 dependent fatty acid hydroxylases. One can distinguish ω-hydroxylases that catalyze the hydroxylation of the terminal methyl of aliphatics acids (ω position) and sub-terminal or in-chain hydroxylases that oxidize carbons in the chain (ω-n position). Since both types of enzymes were discovered about three decades ago, the majority of investigations have focused on the CYP94 and CYP86 families, which mediate ω-hydroxylations. The activities of ω-hydroxylases in cutin synthesis have been clearly established, but the studies of LCR (LACERATA) and att1 (aberrant induction of type three genes), which are the first Arabidopsis thaliana mutants with alterations in coding sequences of CYP86A8 and CYP86A2, show that these types of ω-hydroxylases can be involved in many aspects of plant development. The existence of different ω-hydroxylases in plants with distinct regulation patterns suggests that these enzymes mediate diverse biological processes. Much less information concerning in-chain hydroxylases is available despite the fact that they were initially reported along with ω-hydroxylases. This lack of information might be explained by the very few examples of sub-terminal hydroxy-fatty acids described in plants. We present here the best characterized fatty acid hydroxylases and we discuss their possible roles in plant defense and development, fatty acid catabolism, plant reproduction and detoxification.     

Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids

We assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with fish oil. We also evaluated their effect on lipogenesis and its related enzymes. Rats were administered 100 mg/kg/d fish oil, by oral gavage, for 30 days. The fatty acid profile of total liver lipids was determined by gas–liquid chromatography coupled to mass spectrometry. Individual phospholipid classes and their molecular species were quantified by ESI-MS/MS. Omega 3 fatty acids readily incorporated into hepatic phospholipids, decreased stearoyl-CoA desaturase 16, stearoyl-CoA desaturase, delta 6 desaturase, and delta 5 desaturase activities (calculated as product/substrate ratio) and decreased the “lipogenesis index”, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. Our results show that long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases' activity in the non-steatotic liver. In addition to corroborating advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

Covalently linked heme in cytochrome p4504a fatty acid hydroxylases

Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group.     

Mechanism-based probes of the topology and function of fatty acid hydroxylases.

The use of three mechanism-based probes to investigate the topology and function of fatty acid hydroxylases is discussed. 1) The observation of protein rather than heme alkylation in the reaction of cytochrome P4504A1 with 10-undecynoic acid supports the argument that the enzyme circumvents the inherent preference for omega-1 hydroxylation by restricting access to the ferryl oxygen. 2) The regiochemistry of the ferricyanide-mediated iron-to-nitrogen shift of the cytochrome P450102 (P450BM-3) phenyl-iron complex indicates that the active site of this bacterial fatty acid hydroxylase is open primarily above pyrrole ring A of the prosthetic heme group, 3) Inhibition of clofibrate-mediated peroxisome proliferation in cultured rat hepatocytes by inactivation of cytochrome P4504A1 indicates that omega-hydroxylation of fatty acids provides a signal for peroxisome proliferation.     

Distribution and chemotaxonomic significance of acetylenic fatty acids in mosses of the dicranales

Thirty-eight moss species from four families of the order Dicranales were analysed for the fatty acid composition of their acyl lipids. In the Ditrichaceae and the Dicranaceae numerous species were found to contain acetylenic fatty acids in their triglycerides, 9,12,15-Octadecatrien-6-ynoic acid was the major component, often accounting for more than 80 mol%, whereas 9,12-octadecadien-6-ynoic acid was found in small amounts of less than 5 mol%. In some genera, all the species examined contained acetylenic fatty acids, e.g.Dicranella andDicranum, whereas in the genusCampylopus all five species tested were free of acetylenic compounds. Two genera, Ditrichum andDicranoweisia, were found to have a non-homogeous distribution of acetylenic fatty acids. The chemotaxonomic significance of the fatty acid composition in relation to morphological characters is discussed.     

Synthesis of some acetylenic acids

All of the mono-ynoic acids (excluding both terminal and conjugated acetylenic acids) of chain length C₁₀ to C₁₄ have been prepared. None of these acids show any useful inhibition of the enzyme PG-Synthetase which is used in the synthesis of prostaglandins from arachidonic acid.