Steady-state kinetics of Rhus laccase with rapid substrate

Abstract

Rhus laccase exhibits ping-pong kinetics [1]. Though reductants do not bind, an apparent K m reflecting substrate-independent steps was reported with a DMPD, a rapid substrate. This work examines the effects of pH, D 2O and anions on reductant substrate-dependent (k r) and substrate-independent (k cat) steps. Activity was measured with an O 2 electrode using DMPD as the reductant. The pH dependency of k r is bell-shaped indicating contributions from at least two groups. The group required in its dissociated form has an apparent pk a 7.55 ± 0.12 as reported previously [1], while the group required in its undissociated form has an apparent pk a 8.43 ± 0.23. Anaerobic reduction data does not detect pH-dependencies consistent with these pk a values and forms [2, 3]. In particular, no group with pk a near 7.5 required in its undissociated form is detected. Therefore, the pH-dependency of k r must involve enzymic states specific to catalytic turnover. Both pH-dependent steps are more likely associated with type 2 Cu than type 1 Cu reduction. Type 1 Cu(II) reduction in laccase which has been activated by a reduction-reoxidation cycle does not show these pH-depedencies [4]. A recently derived steady-state rate law implies that this also holds for type 1 reduction during turnover [4]. The pH dependence of k cat is also bell-shaped. The implicated pk a values were: pk a 5.91 ± 0.035 for an acid catalyst and pk a 8.99 ± 0.02 for a base catalyst. Residual activity (0.22 maximal) at high pH, which implies that the putative acid catalyst is not mandatory, was accounted for in the data fits. While k r does not show a solvent isotope effect, k cat does. In 50% D 2O, pH 7.40, k H/k D is 1.36, in 100%, 2.12 ± 0.038. The ratio of the pH independent k cat is 1.48 in 50% D 2O. Thus, proton(s) transfers are implicated in a rate-limiting substrate-independent step. Analyses of the D 2O concentration dependence of k cat at pH 7.4 are consistent with 2 proton transfers. The isotope-exchanging group is most likely functioning as the acid catalyst given the pk a of the base catalyst and the magnitude of the effect at the pH 7.40. Both F − and N 3 − inhibit laccase immediately when they are added during steady-state turnover. The inhibition patterns obtained indicate that both F − and N 3 − inhibit both reductant-dependent and substrate-independent steps. Laccase exhibits partial activity for both the k r and k cat effects when saturated with F −. ESR spectra of laccase at pH 6.0, 4 °C, show that both types 1 and 2 Cu are 30% reduced during steady-state turnover. The concentration of reduced type 1 and 2 are significantly increased when 40 mM F − is added. Stopped-flow experiments show that F − does not affect type 3 reoxidation of O 2 binding. Thus, the ESR results indicate that during steady-state turnover in the presence of F −, the rate-limiting step is a substrate-independent step affecting type 1 and type 2 Cu reoxidation. These results also imply that F − can remain bound to the reduced type 2 Cu.