Identification of the Substrate Access Portal of 5-Lipoxygenase

Abstract

The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase (5-LOX), is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site, thus the point of substrate entry is not known. We asked whether a structural motif, a “cork” present in 5-LOX but absent in other mammalian lipoxygenases, might be ejected to allow substrate access. Our results indicate that reduction of cork volume facilitates access to the active site. However, if cork entry into the site is obstructed, enzyme activity is significantly compromised. The results support a model in which the “cork” that shields the active site in the absence of substrate serves as the active site portal, but the “corking” amino acid Phe-177 plays a critical role in providing a fully functional active site. Thus the more appropriate metaphor for this structural motif is a “twist-and-pour” cap. Additional mutagenesis data are consistent with a role for His-600, deep in the elongated cavity, in positioning the substrate for catalysis.

5‐Lipoxygenase‐activating protein rescues activity of 5‐lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

“…This observation suggests that either these concealed amino acids interact with the lipid bilayer or that the impaired nuclear translocation is a consequence of a conformational change provoked by a mutation that is incompatible with membrane binding. Although we cannot eliminate the second possibility, the same substitutions were not destabilizing in the Stable‐5‐LOX (20). Moreover, there is substantial precedent for membrane anchoring of proteins by the insertion of aromatic amino acid side chains into the bilayer.…”

“…Y181 was additionally replaced by Phe to investigate the possibility that the hydroxyl group of Y181 provides an H bond necessary for catalysis. It has been reported that in the context of Stable‐5‐LOX, neither F177A, Y181A, nor F177/Y181A mutations destabilize the enzyme and do not adversely affect the basal levels of 5‐H (p)ETE production of purified 5‐LOX (in the absence of cellular regulatory cofactors) (20). Expression of 5‐LOX and FLAP in each of the HEK293 cell lines was verified by immunoblotting, with normalization to β‐actin ( Fig .…”

“…Remarkably, we observed that mutations to the corking residues, which are concealed in the active site in the crystal structure, are crucial for 5‐LOX association with the nuclear membrane in the cell. Studies on the purified recombinant 5‐LOX enzyme where the aromatic corking amino acids F177 and Y181 are substituted by Ala demonstrate that removal of the bulky cork does not adversely affect the capacity to oxygenate free AA of Stable‐5‐LOX (20), an engineered 5‐LOX that harbors multiple substitutions to confer stability and remove membrane insertion loops that might promote aggregation (17). Yet in a cellular context, when expressed in the absence of FLAP, these same mutations in 5‐LOX lead to a reduction in the amount of 5‐LOX products analyzed, whether from intact cells or cell homogenates.…”

“…A corked active site has been described for a second LOX (18), and both corked enzymes gain access to their substrates via Ca 2+ ‐dependent membrane binding (19). Recently it has been shown that substitution of the cork forming residues by Ala does not compromise 5‐H(p)ETE production in a cell‐free assay (20). We asked whether these same amino acids that block AA access to the encapsulated active site of 5‐LOX might be important in a cellular context in conjunction with FLAP, perhaps to coordinate membrane binding with unblocking active site access.…”

Phenolic acid phenethylesters and their corresponding ketones: Inhibition of 5‐lipoxygenase and stability in human blood and HepaRG cells

“…CAPE ( 2 ) (−8.8 kcal/mol) formed a hydrogen bond with Leu 420 (OH ··· O: 3.07 Å) and a π‐π interaction with His372. Compound 7 (−8.9 kcal/mol) underwent pi‐pi interactions with His367 and was the only tested ligand that showed a π‐π interaction with Phe177 which may play a part in product specificity . Compounds 9 (−8.7 kcal/mol) and 10 (−8.7 kcal/mol) had pi‐pi interactions with His 372.…”