Substrate Specificity Changes for Human Reticulocyte and Epithelial 15-Lipoxygenases Reveal Allosteric Product Regulation

Autor: Joshua D. Deschamps, Theodore R. Holman, Aaron T. Wecksler, Victor Kenyon
Rok vydání: 2008
Předmět:
Zdroj: Biochemistry. 47:7364-7375
ISSN: 1520-4995
0006-2960
Popis: 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). Scheme 1 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 towards LA (10), while 15-hLO-2 reacts preferentially towards AA (11), although there is a discrepancy in the literature (12) (vida infra). Interestingly, soybean lipoxygenase-1 (sLO-1), a plant homologue and model enzyme of 15-hLO-1, also reacts preferentially towards AA over LA, even though AA is not a native substrate in soybeans (13). The promiscuity in substrate preference for 15-hLO-1 and 15-hLO-2 has relevancy to cellular biology since the products of LA, 13-hydroperoxyoctadienoic acid (13-HPODE), and AA, 15-HPETE, both affect cellular function. For example, in prostate cancer 13-HPODE up-regulates the MAP kinase signaling pathway, which causes cell proliferation and differentiation (14, 15), while 15-HPETE, down-regulates MAP kinase activity and lowers cell proliferation (16), implying a possible role for 15-hLO in cancer progression. The fact that 15-hLO-1 and 15-hLO-2 react with both AA and LA is also interesting in a structural sense because it is unclear exactly how either substrate binds in the active site or how substrate recognition occurs. It has previously been shown for 15-hLO-1 that there are three factors that participate in substrate recognition (17, 18). First, the depth of the active site (F352, I417 and M418) adjusts how far the methyl end of the substrate can enter, dictating which hydrogen atom is abstracted (regio-selectivity). Second, R402 interacts with the carboxylic acid end of the substrate and affects the reactivity with AA and LA equally. This is an intriguing result since it indicates that R402 interacts with both AA and LA, even though the substrates are different in length by two carbons (∼3 A). This suggests that in order to accommodate the different lengths of AA and LA, either a global/local structural change of the enzyme, or a conformational adjustment of the substrate, or both, is required. Finally, F414 appears to facilitate pi-pi stacking with the ω-9 unsaturation, present in both AA and LA. In contrast, little is known about 15-hLO-2 substrate recognition, however, positional specificity has been studied, with D602, V603 and A416 of 15-hLO-2 affecting product distribution of AA products (19, 20). Notably, a comparison of 15-hLO-1 and 15-hLO-2 by sequence alignments indicates that only F352, discussed previously for 15-hLO-1, is conserved in 15-hLO-2, suggesting a possible difference in their molecular mechanism for substrate recognition. The activation specificity of LO is another property which may potentially have relevancy toward the role of LO in cellular function. LO isozymes are inactive in the as-isolated, ferrous state, however, one equivalent of the hydroperoxide product converts the enzyme to the active, ferric state (Scheme 1) (1). Interestingly, the specific LO products which activate particular LO's is quite broad. 5-HPETE, 12-HPETE, 15-HPETE, and 13-HPODE, all have been shown to activate 5-hLO (21, 22), while both 15-HPETE and 13-HPODE have been shown to activate 15-hLO-1 (17) and 15-hLO-2 (12). These data suggest that enzymatic products from one isozyme of LO can be used to activate another LO isozyme, potentially regulating each other's activity in the cell. Allosteric regulation of protein activity is a common phenomenon in biology, and is observed in various LO's. For example, ATP and CaII have been shown to bind to non-catalytic sites in 5-hLO, resulting in the activation of enzyme catalysis (23). For sLO-1, our laboratory has shown that an allosteric site exists, which can be specifically targeted by the allosteric inhibitor, oleyl sulfate (OS), to lower the enzymatic activity (24). In addition, we have observed an increase in the kinetic isotope effect of kcat/Km (Dkcat/Km) for 15-hLO-1 with the addition of OS, further indicating the presence of an allosteric site (24). These experimental results for 15-hLO-1, in conjunction with the other allosteric results for sLO-1 and 5-hLO, led us to surmise that the allosteric site in 15-hLO-1 may have biological relevance and warranted further study. The current investigation utilizes a novel competitive substrate capture method to determine the substrate specificity of both 15-hLO-1 and 15-hLO-2. The use of this method resulted in the discovery that various LO products elicit changes in the substrate specificity for both 15-hLO isozymes and also change the Dkcat/Km for 15-hLO-1, indicating the presence of a product-activated, allosteric regulatory site for both 15-hLO isozymes.
Databáze: OpenAIRE
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