Feb 23, 1996

Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis

The Journal of Biological Chemistry
José Neptuno Rodríguez-LópezRoger N F Thorneley

Abstract

The observed pseudo-first order rate constant for the reaction between a horseradish peroxidase (HRP) variant (R38L)HRPC* and hydrogen peroxide saturates at high peroxide concentrations (Km = 11. 8 mm). The data are consistent with a two-step mechanism involving the formation of an HRP-H2O2 intermediate (k = 1.1 x 10(4) m-1 s-1) whose conversion to compound I is rate-limiting (k = 142 s-1) suggesting that Arg-38 is not only involved in the cleavage of the O-O bond of peroxide but also has an important role in facilitating the rapid binding of H2O2 to HRP. Rapid-scan spectrophotometry revealed the presence of a transient intermediate with a spectrum consistent with a ferric-hydroperoxy complex. At high peroxide concentrations (>500 microM), compound I is converted to compound III without the accumulation of compound II. Spectrophotometric titrations show that arginine 38 is also involved in modulating the apparent affinity of HRPC for reducing substrates such as guaiacol and p-cresol. The spectrum of the complex formed when these substrates bind to the ferric form of the mutant enzyme differs from that observed when they bind to the wild-type ferric enzyme. At neutral and alkaline pH compound I of (R38L)HRPC* was stable and redu...Continue Reading

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Mentioned in this Paper

Arginine hydrochloride
Alpha-Peroxidase
Hydrogen Peroxide
Proteins, Recombinant DNA
Mutagenesis, Site-Directed
Pseudo brand of pseudoephedrine
Peroxidases
Cytokinesis of the Fertilized Ovum
4-cresol, sodium salt
Hormone Refractory Prostate Cancer

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