Temperature-dependent isotope effects in soybean lipoxygenase-1: correlating hydrogen tunneling with protein dynamics.

Abstract

The hydrogen-atom transfer in soybean lipoxygenase-1 (SLO) exhibits a large kinetic isotope effect on k(cat) (KIE = 81) near room temperature and a very weak temperature dependence (E(act) = 2.1 kcal/mol). These properties are consistent with H small middle dot transfer that occurs entirely by a tunneling event. Mutants of SLO were prepared, and the temperature dependence of the KIE was measured, to test for alterations in the tunneling behavior. All mutants studied exhibit KIEs of similar, large magnitude at 30 degrees C, despite an up to 3 orders of magnitude change in k(cat). E(act) for two of the mutants (Leu(754) --> Ala, Leu(546) --> Ala) is larger than for wild-type (WT), and the KIE becomes slightly more temperature dependent. In contrast, Ile(553) --> Ala exhibits k(cat) and E(act) parameters similar to wild-type soybean lipoxygenase-1 (WT-SLO) for protiated substrate; however, the KIE is markedly temperature dependent. The behavior of the former two mutants could reflect increased reorganization energies (lambda), but the behavior of the latter mutant is inconsistent with this description. We have invoked a full H* tunneling model (Kuznetsov, A. M.; Ulstrup, J. Can. J. Chem. 1999, 77, 1085-1096) to explain the temperature dependence of the KIE, which is indicative of the extent to which distance sampling (gating) modulates hydrogen transfer. WT-SLO exhibits a very small E(act) and a nearly temperature-independent KIE, which was modeled as arising from a compressed hydrogen transfer distance with little modulation of the hydrogen transfer distance. The observations on the Leu(754) --> Ala and Leu(546) --> Ala mutants were modeled as arising from a slightly less compressed active site with greater modulation of the hydrogen transfer distance by environmental dynamics. Finally, the observed behavior of the Ile(553) --> Ala mutant indicates a relaxed active site with extensive involvement of gating to facilitate hydrogen transfer. We conclude that WT-SLO has an active site structure that is well organized to support hydrogen tunneling and that mutations perturb structural elements that support hydrogen tunneling. Modest alterations in active site residues increase lambda and/or increase the hydrogen transfer distance, thereby affecting the probability that tunneling can occur. These studies allow the detection and characterization of a protein-gating mode in catalysis.