Distal Site Aspartate Is Essential in the Catalase Activity of Catalase-Peroxidases

Abstract

Structural and biochemical characterization of aspartate 152 at the distal heme side of catalase-peroxidase (KatG) from Synechocystis PCC 6803 reveals an important functional role for this residue. In the wild-type protein, the side chain carboxyl group of Asp152 is 7.8 A apart from the heme iron and is hydrogen-bonded to two water molecules and a KatG-specific large loop. We have prepared the site-specific variants Asp152Asn, Asp152Ser, Asp152Trp, and Pro151Ala. Exchange of Asp152 exhibited dramatic consequences on the bifunctional activity of this unique peroxidase. The turnover number of catalase activity of Asp152Asn is 2.7%, Asp152Ser 5.7%, and Asp152Trp is 0.6% of wild-type activity. By contrast, the peroxidase activity of the Asp152 variants was 2-7 times higher than that of wild-type KatG or Pro151Ala. The KatG-specific pH profile of the catalase activity was completely different in these variants and exchange of Asp152 made it possible to follow the transition of the ferric enzyme to the redox intermediate compound I by hydrogen peroxide spectroscopically and to determine the corresponding bimolecular rate constant to be 7.5 x 10(6) M(-1) s(-1) (pH 7 and 15 degrees C). The reactivity of compound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared with the wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased. A mechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases and involves the distal residues Trp122 and Asp152, is proposed.

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

“…64; www.pymol.org). Asp-137, and even the oxyferryl group in the same manner as suggested for hydrogen peroxide in the catalatic reaction (61). Transformation of diazene to hydrazine and ammonia (Fig.…”

Hydrogen peroxide oxidation by catalase‐peroxidase follows a non‐scrambling mechanism

“…Very similar results within experimental error were obtained with MtbKatG (10 lM) and BLC (10 lM). In any case, two peaks of equal areas for 18 The pH profiles of catalatic activity for KatGs are quite different from those for monofunctional catalases. The latter's catalatic activities are essentially pH-independent from pH 5 to 10 [20], whereas KatGs show a sharp optimum between pH 6 and 7 [9].…”

“…Another important residue at the distal heme cavity is D152, which is part of the substrate channel and has its side chain carboxyl group pointing toward the heme pocket. It participates in maintaining a rigid and extended hydrogen-bond network, which is important for the H 2 O 2 oxidation reaction [18,24]. Variants that had these residues exchanged (D152S, W122F, Y249F, and R439A) showed a significantly reduced catalase (see Table 1) but a normal or even enhanced peroxidase activity [9].…”

Functional Implication Guided by Structure-Based Study on Catalase – Peroxidase (KatG) from Haloarcula Marismortui

“…The covalent adduct has been shown to be generated by autocatalytic process (Ghiladi et al, 2005a). Crystallographic analysis of KatG from Bulkhorderia pseudomallei (Bp) has indicated that the side-chain of R409 (corresponding to HmKatG numbering) shows conformational change depending on both of pH and the physicochemical state of the active site (Carpena et al, 2005 M244L] in BpKatG considerably decreased the catalase activity, whereas the peroxidase activity was even enhanced (Regelsberger et al, 2000;Jakopitsch et al, 2004;Singh et al, 2004 Y218F] in the M244-Y218-W95 covalent adducts induced complete loss of the catalase activity, whereas the peroxidase activity was highly enhanced in Sy KatG (Jakopitsch et al, 2003a;Jakopitsch,et al, 2003b). M244 in the adduct also has any significant role in the enzyme, whereas the substitution of Met to Ile considerably attenuated the catalase activity but did not lose at all, and enhanced the peroxidase activity Singh et al, 2004).…”