Abstract

The broad goals are to compare the iron, reactive methionine and fatty acid binding sites of soybean lipoxygenase and reticulocyte lipoxygenase. Specific approaches include the following. (1) The products of stoichiometric additions of unsaturated fatty acids or their hydroperoxides to lipoxygenase will be determined by HPLC analysis. (2) The iron sites in several soybean lipoxygenase isozymes and in the reticulocyte enzyme will be compared by EPR. (3) The peptide containing an essential, reactive methionine will be isolated and sequenced. (4) The peptide sites of methionine oxidation and alkylation will be compared for soybean and reticulocyte proteins by peptide mapping and amino acid analysis. (5) Proximity of the reactive methionine to other regions of the protein will be examined by photocrosslinking and spin labeling. A new approach for photochemical transfer of fluorescence from methionine to nearby residues will be developed. (6) Photoreactive fatty acids will be examined by steady state kinetics to determine those with affinity for soybean lipoxygenase. Photolysis and proteolysis will be done for appropriate fatty acid derivatives to map the fatty acid binding site. (7) A study will be made of the extent of incorporation of iron isotopes into lipoxygenase by biosynthetic routes in intact reticulocytes. High levels of incorporation would allow additional physical techniques to be used in future studies of the iron environment in lipoxygenase.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036232-04
Application #
3289779
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1986-07-01
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Wu, Fayi; Katsir, Leron J; Seavy, Margaret et al. (2003) Role of radical formation at tyrosine 193 in the allene oxide synthase domain of a lipoxygenase-AOS fusion protein from coral. Biochemistry 42:6871-80
Gaffney, B J; Su, C; Oliw, E H (2001) Assignment of EPR Transitions in a Manganese-Containing Lipoxygenase and Prediction of Local Structure. Appl Magn Reson 21:413-424
Abraham, B D; Sono, M; Boutaud, O et al. (2001) Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron. Biochemistry 40:2251-9
Bowers, C R; Storhaug, V; Webster, C E et al. (1999) Exploring surfaces and cavities in lipoxygenase and other proteins by hyperpolarized xenon-129 NMR. J Am Chem Soc 121:9370-7
Grant, C V; Cope, W; Ball, J A et al. (1999) Electronic Structure of the Aqueous Vanadyl Ion Probed by 9 and 94 GHz EPR and Pulsed ENDOR Spectroscopies and Density Functional Theory Calculations. J Phys Chem B 103:10627-31
Gaffney, B J; Marsh, D (1998) High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes. Proc Natl Acad Sci U S A 95:12940-3
Gaffney, B J; Silverstone, H J (1998) Simulation methods for looping transitions. J Magn Reson 134:57-66
Gaffney, B J; Eaton, G R; Eaton, S S (1998) Electron Spin Relaxation Rates for High-Spin Fe(III) in Iron Transferrin Carbonate and Iron Transferrin Oxalate. J Phys Chem B 102:5536-41
Boyington, J C; Gaffney, B J; Amzel, L M (1997) The three-dimensional structure of soybean lipoxygenase-1: an arachidonic acid 15-lipoxygenase. Adv Exp Med Biol 400A:133-8
Gaffney, B J (1996) Lipoxygenases: structural principles and spectroscopy. Annu Rev Biophys Biomol Struct 25:431-59

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