Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis.
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 reduced to ferric enzyme without apparent formation of compound II upon titration with p-cresol or ascorbic acid, suggesting a change in the rate-limiting step in the peroxidase cycle. Steady-state kinetic analyses carried out at pH 7.0 showed significant increases in the apparent Km for guaiacol, p-cresol, and 2, 2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS). The high stability of the oxyferryl form of (R38L)HRPC* and its low catalytic constant for reducing substrates also shows that arginine 38 modulates the reactivity of HRP compound I.
Rodriguez-Lopez
J N
JN
Nitrogen Fixation Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom. Brighton, BN1 9QG, United Kingdom.
Smith
A T
AT
Thorneley
R N
RN
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003408:Cresols / Q000378:metabolism; D017931:DNA Primers; D005813:Genes, Synthetic; D014644:Genetic Variation; D006139:Guaiacol / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D003408:Cresols; D017931:DNA Primers; D011994:Recombinant Proteins; C032538:4-cresol; D006139:Guaiacol; D001120:Arginine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase