Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

American Chemical Society full text link American Chemical Society Free PMC article
Full text links

Actions

.2008 Jul 15;47(28):7364-75.
doi: 10.1021/bi800550n. Epub 2008 Jun 21.

Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation

Affiliations

Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation

Aaron T Wecksler et al. Biochemistry..

Abstract

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 this paper, we present a novel method for accurately measuring 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 more than 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), affects only the ratio of 15-hLO-1 (more than 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., and 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 be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Comparison of [kcat/Km]AA/[kcat/Km]LA ratio from steady-state kinetics and competitive substrate capture method for 15-hLO-1 and 15-hLO-2 (grey and white, respectively). Dotted line represents no substrate specificity (ratio = 1). Ratio > 1 represents AA specificity; ratio < 1 represents LA specificity. Enzymatic assays were performed in 25 mM Hepes at pH 7.5 at 22 °C.
Figure 2
Figure 2
Competitive substrate capture method saturation curves of [kcat/Km]AA/[kcat/Km]LA versus [Product] (μM) for 15-hLO-1. Data demonstrates similar effects on the substrate specificity for the AA products, 12-HPETE (closed squares) and 12-HETE (open squares), and for the perdeuterated LA products, 13-HPODE (closed circles) and 13-HODE (open circles). Enzymatic assays were performed in 25 mM Hepes at pH 7.5 at 22 °C.
Figure 3
Figure 3
Depiction of the 15-hLO-1 homology model, active site cavity (green), with a docked AA molecule. Selected residues, observed to be catalytically important, are displayed (R402, F414, I417, M418, and the red sphere as the iron).
Figure 4
Figure 4
The 15-hLO-1, homology model cavities from the open form (red) and closed form (green), are superimposed and viewed from two perspectives, with an approximate 90° rotation about the horizontal axis. Note that the closed 15-hLO-1 cavity perspective, on the right side, is the same as that seen in Figure 3. The ferrous ion is represented as a sphere and the cavities were created using Hollow.
Figure 5
Figure 5
The homology model cavities for the closed forms of 15-hLO-1 (green) and 15-hLO-2 (red) are superimposed and viewed from two perspectives, with an approximate 90° rotation about the horizontal axis. Note that the closed 15-hLO-1 cavity perspective, on the right side, is the same as that seen in Figure 3 and 4. The ferrous ion is represented as a sphere and the cavities were created using Hollow.
Scheme 1
Scheme 1
Scheme 2
Scheme 2
See this image and copyright information in PMC

References

    1. Solomon EI, Zhou J, Neese F, Pavel EG. New insights from spectroscopy into the structure/function relationships of lipoxygenases. Chem Biol. 1997;4:795–808. - PubMed
    1. Nakano H, Inoue T, Kawasaki N, Miyataka H, Matsumoto H, Taguchi T, Inagaki N, Nagai H, Satoh T. Synthesis and biological activities of novel antiallergic agents with 5-lipoxygenase inhibiting action. Bioorg Med Chem. 2000;8:373–380. - PubMed
    1. Ghosh J, Myers CE. Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells. Proc Natl Acad Sci U S A. 1998;95:13182–13187. - PMC - PubMed
    1. Steele VE, Holmes CA, Hawk ET, Kopelovich L, Lubet RA, Crowell JA, Sigman CC, Kellof GJ. Lipoxygenase Inhibitors as Potential Cancer Chemopreventives. Cancer Epidemiology, Biomarkers & Prevention. 1999;8:467–483. - PubMed
    1. Hussain H, Shornick LP, Shannon VR, Wilson JD, Funk CD, Pentland AP, Holtzman MJ. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis. Am J Physiol. 1994;266:C243–253. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full text links
American Chemical Society full text link American Chemical Society Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp