Horseradish Peroxidase: Partial Rescue of the His-42 → Ala Mutant by a Concurrent Asn-70 → Asp Mutation

Abstract

In horseradish peroxidase (HRP), hydrogen bonding of Asn-70 to His-42 enhances the catalytic activity of the histidine, and mutation of His-42 to a neutral residue greatly decreases peroxidase activity. The N70D/H42A HRP mutant is compared here to the previously characterized H42A mutant to determine if the Asp-70 substitution can rescue the catalytic activity. The N70D/H42A and H42A mutants give Compound I species with a high ratio of H2O2at the low rates of 37 and 81 M−1s−1at 4°C, respectively. Thekcatvalues for the oxidation of guaiacol and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by N70D/H42A HRP are 2.7 and 143 s−1, respectively, compared to values of 0.015 and 0.41 s−1for the H42A mutant. Thekcatvalues for thioanisole sulfoxidation by the N70D/H42A and H42A mutants are 0.18 and 0.03 s−1, respectively, and the corresponding values for styrene epoxidation are 0.005 and 0.007 s−1. Due to changes in the substrateKmvalues, the efficiencies of the N70D/H42A and H42A mutants defined bykcat/Kmare guaiacol, 5 vs 4; ABTS, 286 vs 68; thioanisole, 3 vs 0.1; and styrene, 0.025 vs 0.002, respectively. The N70D mutation in N70D/H42A HRP thus increases the activity versus the H42A mutant with respect to all four substrates. The increased efficiency is due to enhancements in catalytic steps other than the formation of Compound I.