Kinetic studies of oxygen reactivity in soybean lipoxygenase-1.
The reactivity of O(2) with soybean lipoxygenase-1 (SLO) has been examined using a range of kinetic probes. We are able to rule out diffusional encounter of O(2) with protein, an outer-sphere electron transfer to O(2), and proton transfer as rate-limiting steps in k(cat)/K(M)(O(2)) for wild-type enzyme (WT SLO); this restricts the rate-limiting step to either the combination of O(2) with L(*) or a subsequent conformational change. In the Ile(553) --> Phe mutant, which constricts the putative O(2) binding channel [Knapp et al. (2001) J. Am. Chem. Soc. 123, 2931-2932], k(cat)/K(M)(O(2)) decreases by over a factor of 20; yet, this mutant appears to have the same rate-limiting step as WT SLO. It is argued that the slow step on k(cat)/K(M)(O(2)) is the combination of O(2) with L(*), with proximal protein effects determining the rate of reaction. The available data for SLO support the view that enzymes can affect O(2) reactivity without a direct involvement of metal cofactors. The primary role of the Fe(3+) cofactor is to generate an enzyme-bound radical, while the protein is concluded to control the stereo- and regiochemistry of O(2) encounter with this radical.
D019943:Amino Acid Substitution; D001665:Binding Sites; D003903:Deuterium; D004563:Electrochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008041:Linoleic Acids / Q000494:pharmacology; D008054:Lipid Peroxides / Q000494:pharmacology; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012997:Solvents / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology*; D013237:Stereoisomerism; D014783:Viscosity
D008041:Linoleic Acids; D008054:Lipid Peroxides; D011994:Recombinant Proteins; D012997:Solvents; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D003903:Deuterium; C101084:lipoxygenase L-1; D008084:Lipoxygenase; D010100:Oxygen