INTRODUCTION

Mammalian lipoxygenases have been implicated in the pathogenesis of various inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1, 2). There is also evidence that 15-lipoxygenase may be involved in atherosclerosis. 15-Lipoxygenase is induced in human atherosclerosis lesions (3) and it is capable of oxidizing low density lipoprotein to its atherogenic form (4, 5). All lipoxygenases catalyze the stereo-specific oxygenation of fatty acids containing 1,4-cis,cis-pentadiene. Currently, three distinct mammalian lipoxygenase enzymes have been characterized, 5-, 12-, and 15-lipoxygenases, named for their positional specificity on arachidonic acid. Molecular cloning and sequencing of mammalian lipoxygenases from a variety of organisms have revealed a high degree of sequence identity. Comparison of plant and mammalian lipoxygenases, however, reveals only 25% amino acid identity between the two families (6).

At the time this study was initiated, the only lipoxygenase for which an atomic resolution structure had been determined was soybean lipoxygenase-1 (7, 8). A solvent-accessible cavity within the catalytic domain is proposed to be the substrate binding site (7). Since the structure was solved in the absence of any bound substrate or inhibitor, it is difficult to assign functional relevance to particular structural features. We have chosen site-directed mutagenesis to test hypotheses concerning which amino acids may interact with the substrate. Previous work, using amino acid sequence alignment and site-directed mutagenesis, identified amino acid residues at positions 417 and 418 in the human platelet 12-lipoxygenase and human reticulocyte 15-lipoxygenase (corresponding to sThr⁵⁵⁶ and sPhe⁵⁵⁷ of soybean lipoxygenase-1)¹ as important features of the binding site for the methyl end of the fatty acid substrate (9, 10, 11). Examination of the soybean structure indicates that the homologous residues, sThr⁵⁵⁶ and sPhe⁵⁵⁷, line the internal cavity, providing support that this cavity may be the arachidonic acid binding site in soybean lipoxygenase-1 (7).

We present a model for the binding of arachidonic acid to lipoxygenase based on the high resolution structure of the soybean enzyme (12) and have used this model to identify key amino acid residues in human 15-lipoxygenase that could play a role in substrate binding. Iron is an essential cofactor of the enzyme (13). EPR data have indicated that the iron shuttles from its ferric to its ferrous form during catalysis (14). Thus, the region of the enzyme in proximity to the iron center can be considered the catalytic core of the enzyme. Within a 15-Å sphere of the catalytic core of the enzyme, as revealed by this structure, the sequence identity between the human and soybean enzyme is only 28%. We have, however, assumed that the catalytic core of the plant and mammalian lipoxygenases will be structurally similar, given the similar activities of the enzymes (15). Mutagenesis results validate the assumed structural similarities between the plant and mammalian lipoxygenases and provided support for the proposed arachidonic acid model.

MATERIALS AND METHODS

The program Insight II (Biosym) was used to examine the soybean lipoxygenase-1 structure (12) and to calculate the solvent-accessible surface of the internal cavity (probe size 1.4 Å). Arachidonic acid was manually docked into the proposed substrate binding site, and the quality of the binding modes were visually assessed to determine which amino acid residues could be critical for the proposed substrate binding mode. For these modeling experiments, the protein structure was not modified or adjusted.

Site-directed mutagenesis of human 15-lipoxygenase (6) was performed using the Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad). Oligonucleotides containing the appropriate base changes were synthesized on an Beckman Oligo 1000 DNA synthesizer. Uracil-laden plasmid pSS15LO, used as a mutagenesis template, was prepared as described (16). This plasmid contains a lac promotor to express the 15-lipoxygenase and a bacteriophage f1 origin of replication to generate single-strand DNA. To replace Phe⁴¹⁴ with isoleucine or tryptophan, oligonucleotides with sequence 5-GCTCATTATCTGTCGAAATTCCCATGTC-3 and 5-CCAGTGCTCATTATCTGGTCAAATTCCCATGTCAGAGAC-3 were used respectively (base changes are underlined). The mismatch contained in the first oligonucleotide, T, converted the Phe⁴¹⁴ codon (TTC) to a isoleucine codon (ATC). Another mismatch, A, added a ClaI restriction site to facilitate screening. In the second oligonucleotide, mismatches CC converted the Phe⁴¹⁴ codon to a tryptophan codon (TGG) and introduced an additional AvaII restriction site. To generate Arg⁴⁰²  Leu and Arg⁴⁰²  Lys, primers 5-CCTGGCCGGACGTTATTTCCAGGGTG-3 and 5-CCTGGCCGACGTTATTTCCAGGGTG-3 were used. Mismatches converted codons CGG (Arg⁴⁰²) to CTG (Leu) or AAG (Lys). Mismatch G removed a MseI restriction site. After the mutagenesis reaction, transformants were screened with restriction enzymes. The complete coding region of the mutant cDNAs were sequenced to ensure that there were no second-site mutations.

The Bac-to-Bac^TM baculovirus expression system (Life Technologies, Inc.) was used for the expression of the wild-type human 15-lipoxygenase as well as the mutant proteins described above. A fragment of cDNA containing the complete coding region of human 15-lipoxygenase was cloned into the donor vector pFastBac1 between NotI and SalI sites, to generate the recombinant donor plasmid for wild-type human 15-lipoxygenase. The recombinant donor plasmids for mutant proteins were obtained by replacing the fragment of wild-type cDNA between the two PpuMI sites with the same fragments of each mutated cDNA. This fragment contains the coding region of human 15-lipoxygenase from codon 117 through the stop codon. The recombinant plasmids were transformed into DH10Bac-competent cells containing the bacmid with a mini-attTn7 target site and the helper plasmid. High molecular weight mini-prep DNAs prepared from selected Escherichia coli clones for each engineered protein were used to transfect the Sf9 insect cells and generate the recombinant baculoviruses.

Sf9 cell cultures (1 liter) at a cell density of 2 × 10⁶ cells/ml were infected with each of the recombinant baculoviral stocks (multiplicity of infection of 0.9 plaque-forming units/cell). After 4 days, the cell pellets were collected by centrifugation. All proteins were purified to near homogeneity by high pressure liquid chromatography (17). The iron content of purified enzymes was determined by a colorimetric method using the chromogenic ligand Ferrozine (18).

Lipoxygenase activity was assayed spectrophotometrically (Beckman DU650 spectrometer) by monitoring the increase in absorbance at 234 nm. The standard assay contained 1 ml of 10 mM potassium phosphate buffer (pH 7.0) with 0.2% sodium cholate, 2 µM 13-hydroperoxylinoleic acid (13-HPODE),² and 30 µM linoleate (Biomol) as substrate. The substrate solutions were prepared as described (17). The values of enzyme specific activity were normalized for the iron content of each enzyme.

To estimate the enzyme kinetic parameters of engineered enzymes, assay solutions in the same buffer as above with various substrate concentrations ranging from 5 to 25 uM were used. The reactions were started by addition of 1 µg of purified enzyme and performed in quartz cuvettes at room temperature. The linear part of the reaction curves were used to calculate reaction rates using a molar extinction coefficient of 23,000 M cm¹ for conjugated dienes at 234 nm (19). Values for apparent K_m and V_max were estimated using the Kinetassist program and normalized with iron content of the enzymes.

For each reaction, 5 µg of purified enzyme was added to a substrate solution containing 100 µg of the substrate in 1 ml of 10 mM potassium phosphate buffer (pH = 7.0) and incubated at 37 °C for 15 min. The reaction mixtures were then reduced with trimethyl phosphite and extracted for HPLC analysis as described previously (20). HPLC analyses of the reaction products formed from different substrates were carried out on a Waters 600 HPLC system equipped with a Waters 996 photo-diode array detector. RP-HPLC was performed on a Beckman C-18 column. The column was developed isocratically with methanol/water/acetic acid (80:20:0.1) at a flow rate of 1.5 ml/min, and the column effluent was monitored continuously at wavelengths between 200 and 300 nm. Chiral phase HPLC of the hydroxy fatty acids was carried out on a Chiralcel OD column (Daicel Chemical Industries) using a solvent system of n-hexane/2-propanol/HAc (100:1.5:0.1, v/v/v) and a flow rate of 1.0 ml/min.

Experiments with various amounts of enzyme were performed for the methyl ester substrates. The results showed that the decrease of reactivity toward methyl ester substrates was not caused by the limited solubility of these substrates (data not shown).

RESULTS

The molecular modeling studies reported here utilize the high resolution (1.4 Å) soybean lipoxygenase-1 structure determined by W. Minor and co-workers (12). We generated the solvent-accessible surface for this structure and confirmed the existence of an internal pocket, similar to the pocket identified previously (Fig. 1A) (7, 12, 21). We have focused our attention on the inner cavity as the catalytically relevant binding site for arachidonic acid. This inner cavity contains a broad pocket, which is about 20 Å in length and 6 Å in diameter (Fig. 1B). The catalytic iron is positioned just outside of the solvent-accessible cavity, approximately in the middle, with the coordinating ligands, sHis⁴⁹⁹, sHis⁵⁰⁴, and sIle⁸³⁹, defining part of the cavity. sHis⁶⁹⁰ and sAsn⁶⁹⁴, the other two protein ligands to the iron, do not contact the surface of this cavity. Across from the iron, a narrower pocket, about 3 Å in diameter, extends off the larger pocket at nearly a 90° angle. It is approximately 5 Å from this extension to a narrow neck defined by amino acids sThr⁵⁵⁶ and sPhe⁵⁵⁷. Extending further, the pocket widens into a larger cavity. The end of this larger pocket may open to the outer cavity as described by Amzel and co-workers (7), if the side chains of sArg⁷⁰⁷ and sVal³⁵⁴ adopt slightly different positions. The inner cavity is defined by the 30 amino acid residues shown in Table I.

Table I. Amino acid residues surrounding the proposed arachidonic acid binding site Sequence comparison of soybean lipoxygenase-1 and human 15- and 12-lipoxygenases. Amino acid residues in the solid boxes, bold for the soybean lipoxygenase-1 and bold italic for human 15-lipoxygenase, are suggested by modeling or mutagenesis in this report to be important for substrate binding. Amino acid residues in the dashed boxes were previously suggested to be near the substrate binding site (9, 25). All sequences were accessed from GenBank and compared using the BestFit program in the GCG sequence analysis software package, version 7.3 on a VAX computer.

Docking arachidonic acid into a potential binding site within an enzyme structure is complicated by its conformational flexibility. It has been estimated that arachidonic acid can assume more than 10⁷ low energy conformations (22). Our initial attempts to dock arachidonic acid into the inner cavity of soybean lipoxygenase-1 indicated that this substrate could be placed in the cavity in several different conformations, as well as in several orientations. In an effort to clarify which possible binding mode might be more realistic, assumptions concerning the interaction between the enzyme and arachidonic acid had to be made. First, we assumed that the binding site for the methyl end of arachidonic acid would be defined by sThr⁵⁵⁶ and sPhe⁵⁵⁷, since the homologous amino acid residues in human 15-lipoxygenase, hIle⁴¹⁷ and hMet⁴¹⁸, have been shown to effect positional specificity (9). This would allow arachidonic acid to adopt a conformation that positions the LS-hydrogens (as defined in Ref. 23) of C-10 and C-13 near the catalytic iron.

In developing a possible docking mode for arachidonic acid, we postulated that there may be a positively charged residue in the vicinity of the binding site to interact with the carboxylate group of arachidonic acid. Three positively charged amino acid residues, sLys²⁶⁰, sArg⁷⁰⁷, and sHis⁴⁹⁴, are possible candidates for this interaction based on the soybean lipoxygenase-1 structure. Another possible interaction between the enzyme and the substrate could be that of an aromatic residue with the cis-double bonds of the substrate. In considering various docking orientations of arachidonic acid, the existence of possible -electron interactions was noted.

Arachidonic acid was docked into the inner cavity of soybean lipoxygenase-1 as shown in Fig. 1B. Our working model docked the methyl end of arachidonic acid near the narrow neck of the inner cavity defined by sThr⁵⁵⁶ and sPhe⁵⁵⁷. The carboxylate group of arachidonic acid was docked near sLys²⁶⁰, instead of sArg⁷⁰⁷ or sHis⁴⁹⁴, as this orientation of the substrate allowed the reactive 1,4-cis,cis-pentadiene moiety to be very close to the catalytic iron. This docking mode, therefore, proposes that sLys²⁶⁰ interacts with the carboxylate group of the substrate.

To extrapolate from our model of arachidonic acid binding to the soybean enzyme, we identified conserved mammalian amino acid residues that could play similar roles in the substrate binding site (Table I and Fig. 1B). We identified sLys²⁶⁰ as a potential charged interaction with the substrate. Across the proposed arachidonic acid binding site from sLys²⁶⁰ is sLeu⁵⁴¹. The residue in all mammalian enzymes that corresponds to sLeu⁵⁴¹ is hArg⁴⁰², based on sequence alignments (Figs. 1B and 2). We hypothesized that hArg⁴⁰² could be important for interacting with the carboxylate of the acid substrate. With the substrate modeled in this orientation, sTrp⁵⁰⁰ could form a -electron interaction with arachidonic acid. The interaction postulated for sTrp⁵⁰⁰ and the substrate may be served by hPhe⁴¹⁴ in the mammalian enzymes (Figs. 1B and 2). Another amino acid that was identified from the model was sLeu⁵⁴⁶, which may define the entrance to the narrow pocket occupied by the methyl end of the substrate. The homologous residue in mammalian lipoxygenase is hLeu⁴⁰⁷. It was of interest to investigate the role of this residue in substrate binding, since it is conserved in all of the lipoxygenases. To determine if hArg⁴⁰², hPhe⁴¹⁴, and hLeu⁴⁰⁷ are critical in substrate binding, each was individually replaced by site-directed mutagenesis of human 15-lipoxygenase.

In human 15-lipoxygenase, hArg⁴⁰² may provide a positive charge to interact with the carboxylate group of the substrate and was, therefore, replaced with leucine and lysine. We postulated that hPhe⁴¹⁴ could be involved in an aromatic interaction with the substrate, and it was replaced with isoleucine and tryptophan. hLeu⁴⁰⁷ may be one of the amino acid residues that define the entrance of the narrower pocket of the proposed substrate binding site for the methyl end of the substrate and, therefore, was changed to alanine, isoleucine, and methionine. Preliminary experiments to determine the effects of substituting residues hArg⁴⁰², hPhe⁴¹⁴, and hLeu⁴⁰⁷ were performed using the crude bacteria extracts. All cultures were assessed for protein expression by SDS-polyacrylamide gel electrophoresis, followed by immunoblot detection. To the limits of this technique, the levels of expression were not significantly altered (data not shown). Bacteria expressing the mutated human lipoxygenases were incubated with arachidonic acid. The fatty acid products were extracted and analyzed by RP-HPLC. The activity of both Arg⁴⁰²  Leu and Phe⁴¹⁴  Ile were markedly lower than that of the wild-type enzyme in the crude bacteria extracts (data not shown). Introducing a positively charged residue (Arg⁴⁰²  Lys) or an aromatic residue (Phe⁴¹⁴  Trp) back into the enzyme led to an apparent increase in enzyme activity. The substitutions of hLeu⁴⁰⁷, however, showed no significant effect on either the total enzyme activity or the positional specificity. Based on these preliminary results, mutant proteins relevant to hArg⁴⁰² and hPhe⁴¹⁴ were expressed in the baculovirus/insect cell system and purified to near homogeneity (17). The iron content of the purified mutated protein samples were determined to be similar to that of the wild-type protein.

The steady-state kinetic parameters of the purified mutant enzymes were compared with the wild-type enzyme. Substrate concentrations ranging from 5 to 25 µM were used. The reactions were started by addition of 1 µg of purified enzyme and performed in a quartz cuvette at room temperature. As shown in Table II, all of the mutations altered the values of apparent K_m for fatty acid substrates, while V_max of the mutant enzymes were similar to that of the wild-type enzyme.

Table II. Steady-state kinetic parameters of human 15-lipoxygenase and mutant enzymes The enzyme kinetic parameters of engineered enzymes were performed in 10 mM potassium phosphate buffer (pH 7.0) with 0.2% sodium cholate, 2 µM 13-HPODE. Various substrate concentrations ranging from 5 to 25 µM were used. The reactions were started by addition of 1 µg of purified enzyme and performed at room temperature. The linear part of the reaction curves were used to calculate reaction rates using a molar extinction coefficient of 23,000 M cm1 for conjugated dienes at 234 nm (17). Values for apparent KM and Vmax were estimated using the Kinetassist program and normalized for the iron content of each enzyme (values ± S.E.; n = 3). Lipoxygenase Arachidonic acid Linoleic acid KM Vmaxa KM Cmaxa µM units/mg µM units/mg Wild-type 7.5  ± 0.3 1.03  ± 0.01 7.8  ± 0.5 4.90  ± 0.08 Arg402  Leu 53.5  ± 17.6 1.00  ± 0.01 70.4  ± 9.4 3.28  ± 0.60 Arg402  Lys 23.9  ± 2.6 1.14  ± 0.06 30.9  ± 3.4 4.03  ± 0.67 Phe414  Ile 14.6  ± 9.5 1.00  ± 0.39 17.7  ± 8.5 3.92  ± 0.35 Phe414  Trp 3.6  ± 0.3 1.11  ± 0.13 6.6  ± 1.2 5.31  ± 0.03 a  Unit = µmol of conjugated diene produced/min.

Based on the model, we predicted that replacement of hArg⁴⁰² with leucine would reduce the binding affinity for fatty acid substrates. The activity of Arg⁴⁰²  Leu is about 5% of the wild-type activity against both arachidonic acid and linoleic acid (Table III). Kinetic characterization of Arg⁴⁰²  Leu under steady-state conditions showed that V_max is unaffected, while K_m is increased 7-10-fold depending on the substrate (Table II), consistent with the hypothesis that Arg⁴⁰² is energetically involved in binding the substrate by a charge interaction. Chiral phase HPLC analysis of the enzyme reaction products (15-HETE, 12-HETE, and 13-HODE) indicated that they are predominantly in the S-configuration.

Table III. The role of charged and aromatic residues of human 15-lipoxygenase in substrate binding The enzymatic activities for the native and engineered proteins were assayed spectrophotometrically by monitoring conjugated formation as measured by an increase in absorbance at 234 nm and normalized for the iron content of each enzyme. The standard assay contained 1 ml of 10 mM potassium phosphate buffer (pH 7.0) with 0.2% sodium cholate, 2 µM 13-HPODE, and 30 µM fatty acid as substrate. The product ratios were determined using an HPLC assay. For each reaction, 5 µg of purified enzyme was added to a substrate solution containing 100 µg of the substrate in 1 ml of 10 mM potassium phosphate buffer (pH = 7.0) and incubated at 37 °C for 15 min. RP-HPLC was performed on a Beckman C-18 column; the column was developed isocratically with methanol/water/acetic acid (80:20:0.1) at a flow rate of 1.5 ml/min (values ± S.E.; n = 3). Lipoxygenase Arachidonic acid Linoleic acid (specific activity)a Specific activitya 15-HETE:12-HETEb Wild-type 1.23  ± 0.18 9 :1 5.81  ± 0.06 Arg402  Leu 0.067  ± 0.004 4 :1 0.53  ± 0.14 Arg402  Lys 0.14  ± 0.023 11 :1 1.19  ± 0.08 Phe414  Ile 0.24  ± 0.05 1.6 :1 1.28  ± 0.37 Phe414  Trp 3.23  ± 0.37 25 :1 5.92  ± 0.86 a  Unit = µmol of conjugated diene produced/min/mg of enzyme. b  HETE = hydroxyeicosatetraen-1-oic acid.

Human 15-lipoxygenase has dual positional specificity; oxygenation of arachidonic acid produces two chiral products, 15(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, in a ratio of 10:1. It has previously been suggested that the positional specificity of 15-lipoxygenase is affected by the alignment of a doubly allylic methylene carbon with the catalytic iron of the enzyme (24). To determine the effect of an arginine to leucine substitution at position 402 on the positional specificity, the enzyme reaction products with arachidonic acid as substrate were analyzed by RP-HPLC (Table III). Wild-type human 15-lipoxygenase produced 15-HETE and 12-HETE in a ratio of 10:1 (15-HETE:12-HETE), whereas this ratio was 4:1 for Arg⁴⁰²  Leu. This change in positional specificity implies that hArg⁴⁰² is important in positioning the substrate accurately in the binding site relative to the catalytic iron. When Arg⁴⁰² is replaced with leucine, the substrate apparently can shift in the pocket positioning the C-10 of arachidonic acid closer to the catalytic iron, leading to a relatively higher yield of 12-HETE.

To provide further evidence for the importance of a positive charge in the substrate binding pocket, we constructed the charge-preserving mutation, Arg⁴⁰²  Lys. This amino acid replacement resulted in approximately 2-fold increase in enzyme specific activity when compared to Arg⁴⁰²  Leu, although the activity was 6- and 11-fold lower than that of the wild-type enzyme on arachidonic acid and linoleic acid, respectively (Table III). Importantly, the Arg⁴⁰²  Lys mutation shows the same positional specificity as the wild-type enzyme, which is reflected in the 15-HETE:12-HETE ratio (Table III). pH titration of enzyme specific activity revealed that both wild-type human 15-lipoxygenase and Arg⁴⁰²  Lys mutant have pH optima at 7, while no pH effect on enzyme activity was observed for the Arg⁴⁰²  Leu mutant (data not shown).

The hypothesis that hArg⁴⁰² defines a carboxylic acid binding site predicts that the Arg⁴⁰²  Leu variant would no longer have a preference for acid substrate. We tested this prediction by measuring the activity of the Arg⁴⁰²  Leu enzyme on methyl ester substrate. As shown in Table IV, the activity of wild-type enzyme toward a methyl ester substrate is approximately 10% of that toward the corresponding acid substrates, arachidonic acid and linoleic acid. The Arg⁴⁰²  Leu enzyme has comparable activities for both the acid and methyl ester substrates. Importantly, the Arg⁴⁰²  Leu mutant enzyme is as active as the wild-type enzyme against the methyl ester substrates. These results provide further evidence that the positive charge of Arg⁴⁰² is important for the binding of fatty acid substrates. Furthermore, it shows that replacing the arginine with leucine did not significantly perturb the overall structure of the enzyme beyond the site of substitution.

Table IV. Acid versus methyl ester substrates The relative activities of each enzymes are based on the HPLC analysis of the enzyme reaction products. Assay conditions are the same as described in the legend of Table III. The lower activities against the methyl ester substrates are not due to the limited solubility of the methyl esters, according to the control experiments. Lipoxygenase Relative Activity (%)a Arachidonic acid Arachidonic acid methyl ester Linoleic acid Linoleic acid methyl ester Wild-type 100 7 100 11 Arg402  Leu 7 7 13 9 a  The activity of wild-type human 15-lipoxygenase toward arachidonic acid is about 18% of that toward linoleic acid.

In docking arachidonic acid in the soybean lipoxygenase-1 structure, we hypothesized that an aromatic residue could potentially provide an important -electron interaction with a double bond of the substrate. Extrapolating from the model of arachidonic acid docked in soybean lipoxygenase-1, hPhe⁴¹⁴ is the only aromatic amino acid residue within the predicted substrate binding site of human 15-lipoxygenase that could provide such an interaction. To test this hypothesis, hPhe⁴¹⁴ was replaced with both isoleucine and tryptophan. The Phe⁴¹⁴  Ile mutation markedly decreased enzyme activity, while the Phe⁴¹⁴  Trp mutant behaved essentially like the wild-type enzyme (Table III).

Analysis of the enzyme reaction products reveals that Phe⁴¹⁴  Ile has an increase in 12-lipoxygenase activity relative to 15-lipoxygenase activity (Table III). The loss of the aromatic side chain allows the reactive 1,4-cis,cis-pentadiene moiety of the substrate to shift within the pocket thus producing the altered substrate specificity. The replacement of hPhe⁴¹⁴ with tryptophan resulted in a large enhancement in the enzyme's 15-lipoxygenase activity relative to its 12-lipoxygenase activity (15-HETE:12-HETE = 25:1). Chiral phase HPLC analysis showed there was no change in product configuration (data not shown). Comparison of the kinetic parameters for the enzymes where hPhe⁴¹⁴ has been replaced with isoleucine or tryptophan showed only small changes (Table II).

Using our model of arachidonic acid binding, we could not predict whether the ⁸-, ¹¹-, or ¹⁴-double bond of arachidonic acid would interact with hPhe⁴¹⁴. To identify which, if any, of these three double bonds interacts with hPhe⁴¹⁴, we examined the activity of the enzymes against two other unsaturated fatty acid substrates, which differ in their double bond character. As mentioned above, Phe⁴¹⁴  Ile is less active against arachidonic acid, and the preference for 15-lipoxygenation is lost (15-HETE:12-HETE = 1.6:1) (Table V). Phe⁴¹⁴  Trp is more active than the wild-type enzyme, with very strong preference for 15-lipoxygenase activity (15-HETE:12-HETE = 25:1). Fatty acid 11,14-eicosadienoic acid contains only the ¹¹- and ¹⁴- double bonds. This substrate is, therefore, only available for 15-lipoxygenase activity, producing the product 15-HEDE. Replacement of hPhe⁴¹⁴, with the non-aromatic residue isoleucine again reduced the enzyme activity on this substrate, whereas replacement with tryptophan had little effect on the activity. These results suggest that ⁵- and ⁸-double bond of arachidonic acid is not interacting with the aromatic side chain of hPhe⁴¹⁴.

Table V. Enzyme activity versus the placement of the double bonds of fatty acid substrates To identify which double bond of arachidonic acid interacts with the side chain of Phe414, the enzyme specific activity and positional specificity were determined using fatty acid substrate containing cis-double bonds at different positions. The enzymatic activity for the native and engineered proteins were assayed spectrophotometrically by monitoring conjugated diene formation as measured by an increase in absorbance at 234 nm and normalized with iron content of each enzyme. The standard assay contained 1 ml of 10 mM potassium phosphate buffer (pH 7.0) with 0.2% sodium cholate, 2 mM 13-HPODE, and 30 µM fatty acid as substrate. The product ratios were determined using HPLC assay. For each reaction, 5 µg of purified enzyme was added to a substrate solution containing 100 µg of the substrate in 1 ml of 10 mM potassium phosphate buffer (pH = 7.0) and incubated at 37 °C for 15 min. RP-HPLC was performed on a Beckman C-18 column; the column was developed isocratically with methanol/water/acetic acid (80:20:0.1) at a flow rate of 1.5 ml/min. The position of hydrogen abstraction and oxygenation are indicated for arachidonic acid. , hydrogen abstraction; , oxygenation.

Next, we investigated the activity of these enzymes against the substrate 5,8,11-eicosatrienoic acid, which does not have ¹⁴-double bond and should only produce 12-HETE. Again Phe⁴¹⁴  Ile showed 2-fold lower enzyme activity against this substrate, whereas Phe⁴¹⁴  Trp exhibited virtually unaltered activity compared with the wild-type enzyme. It is, therefore, most likely that the aromatic side chain of hPhe⁴¹⁴ interacts with the ¹¹-double bond of arachidonic acid. Unfortunately, a substrate lacking the ¹¹-double bond (i.e. 5,8,14-eicosatrienoic acid) is not accessible to either 12- or 15-lipoxygenation and, therefore, cannot be tested.

DISCUSSION

Two internal cavities in proximity to the catalytic iron were apparent in the three-dimensional structures of soybean lipoxygenase-1 (7, 21). One of these cavities was initially proposed by Amzel and co-workers to be the arachidonic acid binding site. A similar internal pocket is present in the high resolution soybean lipoxygenase-1 structure used in this study (12). The solvent-accessible surface used in describing this cavity in soybean lipoxygenase-1 was generated in the presence of the polar hydrogen atoms only. If this is the substrate binding site, we can assume that some of the amino acid residues lining the pocket would be repositioned and that the pocket would be larger when occupied by substrate.

Experimental data that support the identification of this inner cavity as the arachidonic acid binding site in soybean lipoxygenase-1 are provided by the results reported here and from previous experiments with 15-lipoxygenase and 12-lipoxygenases (9, 10, 11). Wild-type human 15-lipoxygenase has dual positional specificity, producing both 15-oxygenation and 12-oxygenation products in a ratio of approximately 10:1, from arachidonic acid (17). Earlier experiments showed that the substitution of both hIle⁴¹⁷ and hMet⁴¹⁸ in human 15-lipoxygenase with valine altered the positional specificity of the enzyme to favor 12-lipoxygenation. It was, therefore, suggested that these residues could define a portion of the substrate binding pocket of the enzyme (9, 16). The homologues of the human 15-lipoxygenase residues, hIle⁴¹⁷ and hMet⁴¹⁸, are sThr⁵⁵⁶ and sPhe⁵⁵⁷ in the soybean enzyme (Table I). These residues define a narrow neck of the inner cavity (Fig. 1B). In addition, we have previously shown that hMet⁵⁹⁰ of human 15-lipoxygenase is specifically oxygenated by the enzyme product, 13-HPODE, and hence this residue is also likely to be close to the arachidonic acid binding site (25). The corresponding soybean residue of hMet⁵⁹⁰ is sSer⁷⁴⁷. Examination of the soybean structure indicates that all of these residues, sThr⁵⁵⁶, sPhe⁵⁵⁷, and sSer⁷⁴⁷, line the inner cavity, providing support that this cavity is the arachidonic acid binding site in soybean lipoxygenase-1.

A central feature of our current working model of arachidonic acid binding is a positively charged residue at one end of the cavity (hArg⁴⁰² or sLys²⁶⁰), that interacts with the carboxylate group of the substrate (Fig. 2). Prigge and co-workers also suggested the possibility that sHis⁴⁹⁴ could interact with the carboxylate group of the substrate; however, they were able to rule this out using the results of previously published site-directed mutagenesis experiments (21). We propose that hArg⁴⁰² may interact with arachidonic acid. In agreement with our proposal, enzymatic activity and steady-state kinetics data suggested that replacement of hArg⁴⁰² dramatically affected substrate binding. These results highlight the importance of a positively charged residue in this region of the binding site. Interestingly, this type of interaction has been widely observed in other proteins that bind fatty acids, such as fatty acid transport proteins (26). The change in positional specificity implies that hArg⁴⁰² is also important in positioning the substrate accurately in the binding site relative to the catalytic iron. When hArg⁴⁰² is replaced with leucine, there is no longer a charge interaction; thus, the carboxyl terminus of the substrate is free, enabling the substrate to shift in the pocket. This shift positions the C-10 of arachidonic acid closer to the catalytic iron, leading to a relatively higher yield of 12-HETE. The study with methyl ester substrates provides further evidence that the positive charge of hArg⁴⁰² is important for the binding of fatty acid substrates.

Characterization of Arg⁴⁰²  Lys provides further support for the hypothesis that a positively charged residue is critical for optimal 15-lipoxygenase activity. The fact that Arg⁴⁰²  Lys has reduced activity compared with wild-type enzyme may be attributed to the various structural features that distinguish arginine and lysine. The subtle difference in the electrostatics of arginine and lysine could also contribute to the decrease in enzyme activity. Although both side chains carry the same formal charge, the distribution of charge in an arginine side chain is more delocalized, which may provide a more favorable charge interaction. Taken together with earlier work, our current model places binding determinants at both the methyl and carboxyl termini of the substrate. The absence of a charge interaction would explain the lower activity of the wild-type enzyme on esterified fatty acid.

The studies on Phe⁴¹⁴  Ile and Phe⁴¹⁴  Trp mutant proteins suggest that an aromatic residue near the catalytic iron is also important for enzyme activity and the positioning of the substrate in the correct catalytic register (Fig. 2). In soybean enzyme sTrp⁵⁰⁰, which is analogous to hPhe⁴¹⁴, was modeled to interact with the docked substrate. It is interesting to note that soybean lipoxygenase-1 produces only the 15-lipoxygenase product. Introduction of tryptophan into the substrate binding site of human enzyme creates a stronger preference for 15-lipoxygenation and higher enzyme specific activity, perhaps because of the particular -electron characteristics of this amino acid residue. Interaction of a tryptophan residue with a fatty acid substrate is present in the structure of cellular retinol-binding protein in complex with all-trans-retinol (27). In this case, the aromatic residue is close to the isoprene tail of retinol. The direct interaction between tryptophan and retinol has been confirmed by site-directed mutagenesis and ¹⁹F nuclear magnetic resonance spectroscopy with protein containing ¹⁹F-labeled tryptophan (28, 29).

It is most likely that the aromatic side chain of hPhe⁴¹⁴ interacts with the ¹¹-double bond of arachidonic acid. A recent EPR study of the purple form of soybean lipoxygenase-1 suggested that a reaction intermediate contains an allyl radical delocalized over C-9 through C-11 of linoleic acid (30). A -electron interaction between the substrate and an aromatic amino acid side chain of the enzyme would facilitate the formation of this radical by delocalizing the -electrons. The ⁹-double bond of linoleic acid and the ¹¹-double bond of arachidonic acid are both 6 carbons away from the methyl terminus. Our finding that the ¹¹-double bond of arachidonic acid may participate in a - interaction with the enzyme provides support for the enzyme mechanism proposed by Nelson et al. (30).

Although the catalytic mechanism of lipoxygenases has yet to be fully elucidated, it has been suggested that the rate-limiting step of the reaction is the abstraction of hydrogen, with the help of the oxidized iron center, to form a bis-allelic methylene (31, 32). Molecular oxygen is then inserted stereo-specifically 2 carbons away from the site of hydrogen abstraction, resulting in a hydroperoxy-fatty acid containing a conjugated diene. Studies with stereo-specifically labeled substrates have revealed a large primary kinetic isotope effect and loss of label only when the label is in the pro-S conformation (31, 33, 34, 35). We have docked arachidonic acid into the proposed substrate binding site in an orientation so that both the 10-Ls- and 13-Ls-hydrogens to be positioned close to the catalytic iron and points them directly toward the iron, thereby facilitating hydrogen abstraction.

In summary, we have developed a model for substrate binding in the lipoxygenases and used this model to identify two more amino acid residues in human 15-lipoxygenase that are important substrate binding determinants, hArg⁴⁰² and hPhe⁴¹⁴ (Fig. 2). The model for arachidonic acid binding uses the soybean lipoxygenase-1 structure, whereas the proposed substrate-enzyme interactions were tested and proven using the human enzyme. The structural similarity between these two distantly related enzymes is, therefore, confirmed by these experiments. These results also demonstrate the importance of both a positively charged amino acid residue and an aromatic amino acid residue for fatty acid binding (Fig. 2). Thus, what has been shown previously to be characteristic of non-enzymatic fatty acid-binding proteins is also shown to be an important binding determinant for an enzyme that metabolizes arachidonic acid.