Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.
To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.
Tanaka
M
M
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Ishimori
K
K
Morishima
I
I
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D006735:Horseradish Peroxidase / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008165:Luminol / Q000737:chemistry / Q000378:metabolism*; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D018716:Molecular Mimicry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; D008165:Luminol; D006735:Horseradish Peroxidase; D009195:Peroxidase