Horseradish peroxidase: partial rescue of the His-42 --> Ala mutant by a concurrent Asn-70 --> Asp mutation.
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 H2O2 at the low rates of 37 and 81 M-1 s-1 at 4 degrees C, respectively. The kcat values 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-1 for the H42A mutant. The kcat values 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 substrate Km values, the efficiencies of the N70D/H42A and H42A mutants defined by kcat/Km are 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.
Savenkova
M I
MI
School of Pharmacy, University of California, San Francisco, California, 94143-0446, USA.
Ortiz de Montellano
P R
PR
D052160:Benzothiazoles; D001665:Binding Sites; D002384:Catalysis; D006139:Guaiacol / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D020058:Styrene; D013343:Styrenes / Q000378:metabolism; D013440:Sulfides / Q000378:metabolism; D013451:Sulfonic Acids / Q000378:metabolism
D052160:Benzothiazoles; D013343:Styrenes; D013440:Sulfides; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D020058:Styrene; D006139:Guaiacol; C093850:methylphenylsulfide; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase