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 Fe3+ 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.