2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and methylphenylsulfide

In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.

Topics: Amino Acid Substitution; Amino Acids; Animals; Benzothiazoles; Binding Sites; Catalysis; Ferric Compounds; Heme; Hydrogen Peroxide; Kinetics; Models, Molecular; Myoglobin; Oxidation-Reduction; Peroxidases; Protein Binding; Recombinant Proteins; Spectrophotometry; Stereoisomerism; Sulfides; Sulfonic Acids; Whales

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.

Topics: Benzothiazoles; Binding Sites; Catalysis; Guaiacol; Horseradish Peroxidase; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Mutation; Oxidation-Reduction; Spectrophotometry; Styrene; Styrenes; Sulfides; Sulfonic Acids