Some of the material in is restricted to members of the community. By logging in, you may be able to gain additional access to certain collections or items. If you have questions about access or logging in, please use the form on the Contact Page.
Coral allene oxide synthase (cAOS), a hemoprotein with sequence similarity to bovine liver catalase (BLC), is the N-terminal domain of a fusion protein with an 8R-lipoxygenase in coral Plexaura homomalla. A radical having a g-value of 2.004-2.005 is formed in cAOS upon reaction with peracetic acid. The radical has been shown to be tyrosyl by deuterium substitution of tyrosines and by EPR. The radical site was located at Y193 by mutagenesis of several tyrosines, which are included in the regions of sequence similarity of cAOS and BLC. The kinetics of enzyme reacting with peracetic acid and 13R-HPODE, a nonoptimal substrate, demonstrated that oxidized heme intermediates form and radical formation has a role in recovery of the enzyme. The heme ligand of cAOS was determined to be Y353 by mutagenesis, based on the changed UV-vis spectra of the mutant. Lipoxygenase (LOX) is a family of non-heme iron-containing enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. How LOX interacts with fatty-acid substrates is still not clear although the interaction is critically important in determining the regio- and stereo-specificity of the enzyme. We employed an EPR spectroscopic approach, combined with fatty-acid spin labels, to obtain insight into this question. The fatty-acid spin labels are labeled on carbons 5-, 8-, 10-, 12- and 16- of stearic acid (doxyl stearic acid, DSA). The affinity of DSA for soybean lipoxygenase-1 (LOX-1) increases as the chain length between the labeled carbon and the methyl end increases except in the case of the one labeled on carbon 16. The EPR lineshape suggests that 16-DSA binds to the cavity in a different mode from the others. Iron in LOX-1 enhances relaxation of all the DSA probes similarly. Enzyme kinetics showed that 5-DSA is a competitive inhibitor of LOX-1 with a Ki 9 ?M while the dissociation constant Kd determined by EPR is 10 ?M. These results indicate that all the probes bind to the fatty acid substrate-binding site of LOX-1 or to a portion of it. Room-temperature EPR spectra show local mobility of probes bound to LOX-1. Those probes labeled at the methyl or carboxyl end experienced more motion than those labeled in the middle of the chain. This suggests that the binding site does not accommodate the whole chain, but that doxyl portion resides near the entrance to the substrate cavity, with only the hydrocarbon portion of the spin label occupying the substrate site.