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pubmed:abstractText |
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.
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