Abstract
Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.
Highlights
Solvent isotope effects andthe pH dependenceof data with Rhus vernicifera laccase suggest activaend inactive laccase catalysis under steady-state conditions were formsin theresting enzyme [9,10,11,13,14]
Simulations of the p(H,D) data suggested thatmolecular steps usually cannot beexamined by transient the solvent isotope effect was associated with the pro- kinetic methods; the rate of the reaction is limited by the tein protic group required in its undissociated form relatively slow rate of intermolecular electron transfer
The pH effects on reductant-dependent steps without fast enough reductants, steady-state kinetic experiareapparentlyassociated with reductant-dependent ments seem to offer the best approach for investigatingintrasteps that occur between O2binding andwater forma- molecular steps in laccase catalysis
Summary
Solvent isotope effects andthe pH dependenceof data with Rhus vernicifera laccase suggest activaend inactive laccase catalysis under steady-state conditions were formsin theresting enzyme [9,10,11,13,14]. A significantsolvent isotopeeffect on a reductantindependent step(s) was observed; kdkD = 2.12 at the Earlier steady-state kinetic studies showed that laccase, in the presence of relatively slow reductants, did not exhibit rate saturation [22]. Under these conditions, no apparent K,,, for reductants was observed, whichis consistent with reductants not binding to laccase.When rapid substrates wereused, saturation kinetics were observed [22].Saturation was attributed to a rate-limiting intramolecular step. An extensive characterization of Rhus laccase steady-state kinetics in the presence of rapid substrates is reported here
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