Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In the current paper, we present a novel method to accurately measure the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the [k cat /K m ] AA /[k cat /K m ] LA ratio over 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-(S)-hydroperoxyeicosatetraenoic acid (12-HPETE), only affects the ratio of 15-hLO-1 (over 5-fold). In addition, the reduced products, 13-(S)-hydroxyoctadecadienoic acid (13-HODE) and 12-(S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the [k cat /K m ] AA /[k cat /K m ] LA ratio. These results, coupled with the dependence of the 15-hLO-1, k cat /K m kinetic isotope effect ( D k cat /K m ) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may have relevancy to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.Human lipoxygenases (hLO) are a family of structurally related enzymes that catalyze the hydroperoxidation of polyunsaturated fatty acids using molecular oxygen (Scheme 1) (1). There are three main isozymes of pharmacological interest: 5-hLO, 12-hLO and 15-hLO, which are named according to their positional specificity on arachidonic acid (AA), producing their respective hydroperoxyeicosatetraenoic acid (HPETE) products. Each of these lipoxygenase isozymes plays a distinct biological role in human disease; 5-hLO is implicated in asthma (2) and cancer (3,4), 12-hLO is implicated in psoriasis (5) and cancer (4,6,7) and 15-hLO is implicated in atherosclerosis (8) and cancer (4,9).Defining the exact role of LO in human disease is complicated by the incomplete understanding of three fundamental biochemical properties of hLO: substrate specificity, activation specificity and allosteric regulation. With regards to substrate specificity, enzymes are typically highly specific and react with only one particular substrate, such as 5-hLO and platelet 12-hLO, which only react with AA. However, reticulocyte 15-hLO-1 and epithelial 15-hLO-2 react with both LA and AA, albeit with different efficiencies. 15-hLO-1 reacts preferentially $ This work was supported by the National Institutes of Health (GM56062 and S10-RR20939 (MS equipment grant)) *To whom the correspondence should be addressed: tholman@chemistry.ucsc.edu. % Current address, KaloBios, 3427 Hillview Ave., Sui...

Abstract. Lipoxygenases (LO) have been implicated in asthma, immune disorders, and various cancers and as a consequence, there is great interest in isolating selective LO isozyme inhibitors.Currently, there is much use of baicalein as a selective human platelet 12-LO (12-hLO) inhibitor however, our current steady-state inhibition data indicates that baicalein is not selective against 12-hLO versus human reticulocyte 15-LO-1 (15-hLO-1) (15/12 = 1.3), in vitro. However, in the presence of detergents baicalein is slightly more selective (15/12 = 7), which may imply greater selectivity in a cell based assay but has yet to be proven. The mechanism of baicalein inhibition of 15-hLO is reductive, which computer docking suggests is through direct binding of the catecholic moiety of baicalein to the iron. A structurally related flavonoid, apigenin, is not reductive, however, computer docking suggests a hydrogen bond with Thr591, may account for its inhibitor potency.

Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. The Dkcat/KM for 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates. For AA, there are multiple rate determining steps (RDS) at both high and low temperature, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), however, no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (kcat/KM)AA/(kcat/KM)LA ratio was observed to have a pH dependence, which was fit with a titration curve (pKa = 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizing d31-LA as an inhibitor, it was demonstrated that the binding of d31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (kcat/KM)AA/(kcat/KM)LA ratio. Finally, the magnitude of the 13-HODE KD for 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9, which would alter the LO product distribution by increasing the production of the pro-tumorigenic 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

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Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors from the NCI repository. One is the potent but non-selective michellamine B, a natural product, antiviral agent. The other four compounds were selective inhibitors against 12-hLO, with three being synthetic compounds and one being α-mangostin, a natural product, caspase-3 pathway inhibitor. In addition, a selective inhibitor was isolated from the UCSC-MEL (neodysidenin), which has a unique chemical scaffold for an hLO inhibitor. Due to the unique structure of neodysidenin, steady-state inhibition kinetics were performed and its mode of inhibition against 12-hLO was determined to be competitive (K i = 17 µM) and selective over reticulocyte 15-hLO-1 (K i 15-hLO-1/12-hLO > 30).