PROBING THE ACTIVE-SITE RESIDUES IN AROMATIC DONOR OXIDATION IN HORSERADISH-PEROXIDASE - INVOLVEMENT OF AN ARGININE AND A TYROSINE RESIDUE IN AROMATIC DONOR BINDING

The plausible role of arginine and tyrosine residues at the active sit e of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidati on was probed by chemical modification followed by characterization of the modified enzyme, The arginine-specific reagents phenylglyoxal (PG O), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzym e, following pseudo-first-order kinetics with second-order rate consta nts of 24 M(-1). min(-1), 0.8 M(-1). min(-1) and 0.54 M(-1). min(-1) r espectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first- order kinetics with a second-order rate constant of 2.0 M(-1). min(-1) . The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. The se studies indicate the involvement of arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxa I-modified enzyme showed almost the same binding parameter (K-d) as th e native enzyme, and a similar free energy change (Delta G') for the b inding of the donor. Stoicheiometric studies with [7-C-14]phenylglyoxa l showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, in dicating modification of one arginine residue for complete inactivatio n. The difference absorption spectrum of the tetranitromethane-modifie d against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. I nactivation stoicheiometry showed that modification of one tyrosine re sidue per enzyme caused 50% inactivation. Binding studies by optical d ifference spectroscopy indicated that the arginine-modified enzyme cou ld not bind guaiacol at all, whereas the tyrosine-modified enzyme boun d it with reduced affinity (K-d 35 mM compared with 10 mM for the nati ve enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher t han native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, o wing to lack of binding. No non-specific change in protein structure d ue to modification was evident from circular dichroism studies. We the refore suggest that the active site of HRP for aromatic donor oxidatio n is composed of an arginine and an adjacent tyrosine residue, of whic h the former plays an obligatory role in aromatic donor binding wherea s the latter residue plays a facilitatory role, presumably by hydropho bic interaction or hydrogen bonding.