In mammals, the hydroperoxidation of arachidonic acid by lipoxygenases leads to the formation of leukotrienes and lipoxins, compounds that mediate inflammatory responses. Lipoxygenases are dioxygenases that contain a nonheme iron and are present in many animal cells. Soybean lipoxygenase-1 is a single-chain, 839-residue protein closely related to mammalian lipoxygenases. The structure of soybean lipoxygenase-1 solved to 2.6 angstrom resolution shows that the enzyme has two domains: a 146-residue beta barrel and a 693-residue helical bundle. The iron atom is in the center of the larger domain and is coordinated by three histidines and the COO- of the carboxyl terminus. The coordination geometry is nonregular and appears to be a distorted octahedron in which two adjacent positions are not occupied by ligands. Two cavities, in the shapes of a bent cylinder and a frustum, connect the unoccupied positions to the surface of the enzyme. The iron, with two adjacent and unoccupied positions, is poised to interact with the 1,4-diene system of the substrate and with molecular oxygen during catalysis.

A third phase transition, centered at about 18'C, was observed by differential scanning calorimetry in a multilamellar suspension of dipalmitoyl phosphatidyicholine that had been held at 0WC for several days. This transition is less cooperative than the other two transitions which are well Inown for this system, and it is accompanied by an enthalpy increase about three times as large as that of the so-called pretransition at 350C and about half that of the main gel to liquid crystal transition at 41'C. The reversal of this transition on cooling is extremely slow. The physical properties of phosphatidylcholine bilayers suspended in excess water have been investigated extensively largely because these structures serve as model systems for biological membranes. The two thermotropic phase changes in multilamellar suspensions of dipalmitoyl phosphatidylcholine (Pam2-PtdCho) and related lipids are now well-characterized phenomena (1). In addition to the gel to liquid crystalline phase transition (the "main transition," tm z 41'C for Pam2-PtdCho), a second, broader phase change or "pretransition" occurs at a lower temperature (tmnt 350C for Pam2-PtdCho). The nature of this latter transition is now well understood. In this communication we report the discovery and study by high-sensitivity differential scanning calorimetry (DSC) (2) of a third endothermic phase change in Pam2-PtdCho bilayers, which occurs at a temperature (tom -18"C) below that of the pretransition. In this report we will refer to this transition as the "subtransition.'" MATERIALS AND METHODS Pam2-PtdCho of very high purity (99.94 mol %) was a gift from N. Albon (3). Commercial grade dimyristoyl phosphatidylcholine (Myr2-PtdCho), Pam2-PtdCho, and distearoyl phosphatidylcholine (Ste2-PtdCho) were purchased from Sigma and Calbiochem; dipentadecanoyl phosphatidylcholine [(C15)2-PtdCho] and diheptadecanoyl phosphatidylcholine [(C17)2-PtdCho] were obtained from Supelco (Bellefonte, PA). Liposomes were prepared by adding the appropriate amount of 20 mM sodium phosphate buffer, pH 7.4, to the dry lipid to give a lipid concentration of 0.5-1.0 mg ml-l, heating the mixture to a few degrees above the main transition temperature for 1 hr, and then shaking on a Vortex mixer for several minutes. The lipid suspensions were cooled to 00C for various periods of time before being loaded into the precooled calorimeter. IdenticalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. results were obtained with liposomes prepared from lipid that had been dried down from a chloroform solution or suspended in 10 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer, pH 7.4.All calorimetric scans were made with the Privalov DSC (4), with a scan rate usually of 0.5 K min-, occasionally of 1 or 0.1 K min-1. After the DSC experiments the lipids were extracted and examined by thin-layer chromatography. In no case was ...

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff N 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff E 6.7-5.4 region correlates with the activity of the enzyme. The I n our attempts to understand the mechanism of action of phenylalanine hydroxylase (PAH) from rat liver, we (Gottschall et al., 1982) recently confirmed and extended an earlier observation by Fisher et al. (1972) that a tightly bound non-heme iron was necessary for the activity of the enzyme. We demonstrated that (1) the correct stoichiometry is one atom per subunit, (2) the enzymatic activity is proportional to the iron content, and (3) the iron can be removed from the enzyme and restored with nearly complete recovery of initial activity. In this paper we corroborate and expand the independent discovery (Marota & Shiman, 1984) that PAH is initially reduced to the catalytically active enzyme by the concomitant oxidation of 6-methyltetrahydropterin (6MPH4) directly to the quinonoid form in a stoichiometric reaction requiring oxygen but without the formation of reduced forms of oxygen such as superoxide and hydrogen peroxide, or of tyrosine. In this paper we (1) propose a two-step kinetic sequence for PAH activation and its catalytic turnover based on stopped-flow kinetic and complimentary product data, (2) define by EPR that the resting state of the activatable enzyme is associated with signals observed at geff = 6.7 and 5.4 consistent with high-spin Fe3+ (S = (3) show that the additional EPR signal observed at geff = 4.3 is associated with a form of the enzyme that is incapable of turnover, and (4) link the prereduction step to the conversion of PAH from an Fe3+ to an Fez+ state. Finally, we demonstrate that dithionite can substitute for 6MPH4 in the prereduction step and that the addition of one electron/subunit is sufficient to impart tightly coupled turnover. Experimental Procedures MaterialsDoubly distilled deionized water was used throughout. All reagents were of the highest grade commercially available. 0006-2960/84/0423-1295%01 SO10latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of ophenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fez+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing ...

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3 Hand 11S-3 H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S-and 8S,15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R,15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

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