Probing the Active Site Residues in Aromatic Donor Oxidation in Horseradish Peroxidase : Involvement of an Arginine and a Tyrosine Residue in Aromatic Donor Binding
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Probing the Active Site Residues in Aromatic Donor Oxidation in Horseradish Peroxidase : Involvement of an Arginine and a Tyrosine Residue in Aromatic Donor Binding
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| Creator |
Adak, Subrata
Majumder, Avijit Banerjee, Ranajit K |
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| Subject |
Structural Biology & Bioinformatics
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| Description |
The plausible role of arginine and tyrosine residues at the active site of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modiÆcation followed by characterization of the modiÆed enzyme. The arginine-speciÆc reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-Ærstorder kinetics with second-order rate constants of 24 M−"[min−", 0.8 M−"[min−" and 0.54 M−"[min−" respectively. ModiÆcation with tetranitromethane, a tyrosine-speciÆc reagent, also resulted
in 50%loss of activity following pseudo-Ærst-order kinetics with a second-order rate constant of 2.0 M−"[min−". The substrate, H#O#, and electron donors such as I− and SCN− oåered no protection against inactivation by both types of modiÆer, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the
enzyme. These studies indicate the involvement of arginine and
tyrosine residues at the aromatic donor site of HRP. The guaiacolprotected
phenylglyoxal-modiÆed enzyme showed almost the
same binding parameter (Kd) as the native enzyme, and a similar
free energy change (DG´) for the binding of the donor. Stoicheiometric
studies with [7-"%C]phenylglyoxal showed incorporation
of 2 mol of phenylglyoxal per mol of enzyme, indicating
modiÆcation of one arginine residue for complete inactivation.
The diåerence absorption spectrum of the tetranitromethanemodiÆed
against the native enzyme showed a peak at 428 nm,
characteristic of the nitrotyrosyl residue, that was abolished by
treatment with sodium dithionite, indicating speciÆc modiÆcation
of a tyrosine residue. Inactivation stoicheiometry showed that
modiÆcation of one tyrosine residue per enzyme caused 50%
inactivation. Binding studies by optical diåerence spectroscopy
indicated that the arginine-modiÆed enzyme could not bind
guaiacol at all, whereas the tyrosine-modiÆed enzyme bound it
with reduced aænity (Kd 35 mM compared with 10 mM for the
native enzyme). Both the modiÆed enzymes, however, retained
the property of the formation of compound II (one-electron
oxidation state higher than native ferriperoxidase) with H#O#,
but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modiÆed enzyme, owing to lack of binding. No non-speciÆc change in protein structure due to modiÆcation was evident from circular dichroism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.
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| Date |
1996
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| Type |
Article
PeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/1031/1/bioJaromatic.pdf
Adak, Subrata and Majumder, Avijit and Banerjee, Ranajit K (1996) Probing the Active Site Residues in Aromatic Donor Oxidation in Horseradish Peroxidase : Involvement of an Arginine and a Tyrosine Residue in Aromatic Donor Binding. Biochemical Journal, 314. pp. 985-991. |
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| Relation |
http://dx.doi.org/
http://www.eprints.iicb.res.in/1031/ |
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