Studies of fatty acid metabolism using hydrogen isotopes
Part I. Stereochemistry of the Lipoxygenase Reaction
Lipoxygenases are enzymes that catalyze the stereospecific introduction of molecular oxygen at a terminal carbon of the 1(Z),4(Z)-pentadienyl partial structure of naturally occurring polyunsaturated fatty acids:
The absolute configuration of hydroperoxides generated by most lipoxygenases is "S" as was first shown for soybean lipoxygenase-1 (1). However, exceptions are known, and recently Coffa and Brash (2) described an interesting relationship between product stereochemistry and a specific amino acid residue present in the catalytic domain of lipoxygenases. This amino acid was found to be conserved as an Ala in S lipoxygenases and as a Gly in R lipoxygenases.
As seen above, one of the two hydrogens of the bis-allylic C-3 methylene group is eliminated from the fatty acid substrate during the lipoxygenase-catalyzed formation of a hydroperoxide. According to the "three-point combination concept" advanced by Ogston in 1948 (3) it seemed likely already in early studies of lipoxygenase that this enzyme should discriminate between the two bis-allylic hydrogens, i.e. one of the two should be lost and the other retained. Experimental testing of this hypothesis required the availability of a lipoxygenase substrate stereospecifically labeled at the bis-allylic methylene group with deuterium or tritium. Hamberg and Samuelsson in 1967 (1) described a synthesis of 8(Z),11(Z),14(Z)-eicosatrienoic acid labeled with tritium in the 13(S) positions. Key features in the synthesis was the preparation of 11(R)-hydroxystearate of >99% optical purity, conversion of this compound into its p-toluenesulfonate ester with complete retention of configuration, and lithium aluminium tritiide reduction of the 11(R)-tosylate to introduce the tritium label. Such reductions proceed with inversion of absolute configuration and are accompanied by insignificant racemization. By inverting the configuration of the 11(R)-hydroxystearate prior to the reduction step, the enantiomeric [13(R)-3H]8,11,14-eicosatrienoic acid could also be prepared. The two specimens were mixed with [3-14C]8,11,14-eicosatrienoic acid and the enrichment of tritium in precursors and products could be easily determined by liquid scintillation counting.
Scheme 1. Synthesis of [13(S)-3H]8,11,14-eicosatrienoic acid starting with 3(R,S)-hydroxydecanoic acid. Reactions: i, resolution of the cinchonidine salt; ii, acetylation; iii, anodic coupling with methyl hydrogen sebacate; iv, OH-; v, CH2N2; vi, TsCl/py; vii, LiAl3H4; viii, CrO3; ix, microbial desaturation; x, malonic ester synthesis.
Incubation of the two stereospecifically labeled eicosatrienoates with soybean lipoxygenase-1 afforded 15(S)-hydroperoxyeicosatrienoates which retained the tritium label when formed from the 13(R)-tritio acid but lost the label when formed from the 13(S)-tritio acid.
Scheme 2. Incubation of stereospecifically tritiated 8,11,14-eicosatrienoic acids with soybean lipoxygenase-1.
Assuming that the fatty acid chain adopts its preferred, extended conformation, the results mentioned demonstrated that there is an antarafacial relationship between hydrogen abstraction and oxygen insertion in the reaction catalyzed by soybean lipoxygenase-1:
Subsequent stereochemical studies of the lipoxygenase reaction using a number of lipoxygenases from plants (4-5) and animals (6-13) have invariably given the same result, i.e., hydrogen elimination and oxygen addition take place on different sides with respect to the plane of the Z,Z-pentadiene system.
An unusual case is the recently described manganese-lipoxygenase from the fungus Gaumannomyces graminis (14). Here linoleic acid is converted into two isomeric hydroperoxides, i.e. the bis-allylic 11(S)-hydroperoxylinoleic acid and the allylic 13(R)-hydroperoxyoctadecadienoic acid. Experiments with 11(R)- and 11(S)-deuterio-linoleic acids have demonstrated that biosynthesis of both hydroperoxides takes place with loss of the pro-S hydrogen from C-11 and retention of the pro-R hydrogen. Accordingly, the 11(S)-hydroperoxide is formed by direct substitution at C-11 with retention of absolute configuration, whereas the 13(R)-hydroperoxide is formed with suprafacial stereochemistry. Interestingly, a suprafacial rearrangement of the 11(S)-hydroperoxide into the 13(R)-hydroperoxide was also documented for this enzyme:
Myoglobin has been studied as a lipoxygenase model (15). In this case, the protoporphyrin-FeIII of metmyoglobin was oxidized with hydrogen peroxide into the protoporphyrin-FeIV=O species, which in the presence of molecular oxygen converted linoleic acid into mainly 9(S)-hydroperoxy-10,12-octadecadienoic acid. Incubation of 11(R)- and 11(S)-deuterio-linoleates with this system afforded 9(S)-hydroperoxide which lost and retained, respectively the deuterium label. In other words, this quasi-lipoxygenase reaction proceeded in an antarafacial way like true lipoxygenase reactions.
The lipoxygenase reaction proceeds by way of a carbon-centered pentadienyl radical and a peroxyl radical and has many features in common with autoxidation and other nonenzymatic lipid peroxidations. It is well known that such processes proceed with random regiochemistry (each 1(Z),4(Z)-pentadiene system of a given fatty acid produces comparable amounts of 1- and 5-hydroperoxides) and without stereocontrol (the hydroperoxides produced are racemic). However, a hidden stereospecificity with either an antarafacial or a suprafacial relationship between hydrogen abstraction and oxygen insertion was still conceivable for nonenzymatic oxygenations. This problem was studied by Brash and coworkers (16), who subjected [10(R)-3H]- and [10(S)-3H]arachidonates to autoxidation and separated the individual oxygenated products in enantiomerically pure form. Determination of the isotope contents of the isolated 8(R)- and 8(S)-hydroxyeicosatetraenoates showed identical levels in both enantiomers (40.5-42.5% in different experiments), thus demonstrating that hydrogen loss bears no stereochemical relationship to oxygen insertion in autoxidation.
References
1. Hamberg, M., and Samuelsson, B. (1967) On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase. J. Biol. Chem. 242, 5329- 5335.
2. Coffa, G., and Brash, A.R. (2004) A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation. Proc. Natl. Acad. Sci. USA 101, 15559-15584.
3. Ogston, A.G. (1948) Interpretation of experiments on metabolic processes, using isotopic tracer elements. Nature 162, 963.
4. Egmond, M.R., Vliegenthart, J.F.G., and Boldingh, J. (1972) Stereospecificity of the hydrogen abstraction at carbon atom n-8 in the oxygenation of linoleic acid by lipoxygenases from corn germ and soya beans. Biochem. Biophys. Res. Commun. 48, 1055-1060.
5. Hamberg, M., and Gerwick, W.H. (1993) Biosynthesis of vicinal dihydroxy fatty acids in the red alga Gracilariopsis lemaneiformis: identification of a sodium-dependent 12-lipoxygenase and a hydroperoxide isomerase. Arch. Biochem. Biophys. 305, 115- 122.
6. Hamberg, M., and Hamberg, G. (1980) On the mechanism of the oxygenation of arachidonic acid by human platelet lipoxygenase. Biochem. Biophys. Res. Commun. 95, 1090-1097.
7. Maas, R.L., and Brash, A.R. (1983) Evidence for a lipoxygenase mechanism in the biosynthesis of epoxide and dihydroxy leukotrienes from 15(S)- hydroperoxyeicosatetraenoic acid by human platelets and porcine leukocytes. Proc. Natl. Acad. Sci. USA 80, 2884-2888.
8. Corey, E.J., and Lansbury, P.T., Jr. (1983) Stereochemical course of 5-lipoxygenation of arachidonate by rat basophil leukemic cell (RBL-1) and potato enzymes. J. Amer. Chem. Soc. 105, 4093-4094.
9. Brash, A.R., Ingram, C.D., and Maas, R.L. (1986) A secondary isotope effect in the lipoxygenase reaction. Biochim. Biophys. Acta 875, 256-261.
10. Hawkins, D.J., and Brash, A.R. (1987) Eggs of the sea urchin, Strongylocentrotus purpuratus, contain a prominent (11R) and (12R) lipoxygenase activity. J. Biol. Chem. 262, 7629-7634.
11. Brash, A.R., Yokoyama, C., Oates, J.A., and Yamamoto, S. (1989) Mechanistic studies of the dioxygenase and leukotriene synthase activities of the porcine leukocyte 12S- lipoxygenase. Arch. Biochem. Biophys. 273, 414-422.
12. Hughes, M.A., and Brash, A.R. (1991) Investigation of the mechanism of biosynthesis of 8-hydroxyeicosatetraenoic acid in mouse skin. Biochim. Biophys. Acta 1081, 347- 354.
13. Schneider, C., Keeney, D.S., Boeglin, W.E., and Brash, A.R. (2001) Detection and cellular localization of 12R-lipoxygenase in human tonsils. Arch. Biochem. Biophys. 386, 268-274.
14. Hamberg, M., Su, C., and Oliw, E. (1998) Manganese lipoxygenase: discovery of a bis- allylic hydroperoxide as product and intermediate in a lipoxygenase reaction. J. Biol. Chem. 273, 13080-13088.
15. Rao, S.I., Wilks, A., Hamberg, M., and Ortiz de Montellano, P.R. (1994) The lipoxygenase activity of myoglobin: oxidation of linoleic acid by the ferryl oxygen rather than protein radical. J. Biol. Chem. 269, 7210-7216.
16. Brash, A.R., Porter, A.T., and Maas, R.L. (1985) Investigation of the selectivity of hydrogen abstraction in the nonenzymatic formation of hydroxyeicosatetraenoic acids and leukotrienes by autoxidation. J. Biol. Chem. 260, 4210-4216.