The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1 : 1 stoichiometry and is characterised by an equilibrium dissociation binding constant, Kd, of 11.6 t 0.4 pM (pH 7.002, p = 0.10 M, 25.0OC). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60 %) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM- Keywords: heme; peroxidase; ascorbate peroxidase; binding site.Haem peroxidases comprise a family of metalloenzymes that catalyse the H,O,-dependent oxidation of a wide range of organic and inorganic substrates (Frew and Jones, 1984;Everse et al., 1991 ;Welinder, 1992a;Poulos, 1993;English, 1994; English and Tsaprailis, 1995;Traylor and Traylor, 1995). The first published crystal structure for a haem peroxidase was that of cytochrome-c peroxidase (CcP) (Finzel and Poulos, 1984). More recently, structural information has been published for other peroxidases, namely manganese peroxidase (MnP), lignin peroxidase, peanut peroxidase, myeloperoxidase and Arthromyces ra-~O S U S peroxidase (which is identical to Coprinus cinereus peroxidase) (Sundaramoorthy et al., 1994;Poulos et al., 1993;Schuller et al., 1996;Zeng and Fenna, 1992;Petersen et al., 1994; Kunishima et al., 1994). Detailed comparison of the active-site structures of these enzymes has revealed a high level of sequence similarity around the haem, with five of the nine invariant residues in the plant peroxidase superfamily (Welinder, 1992b) forming part of the active site. Furthermore, site-directed-mutagenesis studies involving active-site residues, predominantly with CcP (Mauro et al., 1989;Erman et al., 1992Erman et al., , 1993Vitello et al., 1992Vitello et al., , 1993 Ferrer et al., 1994 (Smith et al., 1992a(Smith et al., , b, 1993Sanders et al., 1994; Miller et al., 1995;Newmeyer and Ortiz de Montellano, 1995, 1996; Ozaki and Ortiz de Montellano, 1995 ;Neptuno Rodriguez-Lopez et al., 1996), have helped to provide comprehensive understanding of the catalysis at the amino acid level.However, despite an increasingly detailed understanding of the mechanistic aspects of peroxidase catalysis at the active site, there is a very limited understanding of the way in which reducing substrates, in particular small organic substrates, are bound and oxidised by the enzyme. The deficiency arises, at least in part, from the absence of structural information for p...
The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-accesschannel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (8-meso phenyl) and C1 8-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03 % of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkably constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.Keywords: horseradish peroxidase; suicide inactivation; phenylhydrazine; site-directed mutagenesis.Classical peroxidases, including horseradish peroxidase (HRP), have been shown to catalyse the transfer of electrons in two distinct one-electron steps from a wide range of aromatic donor molecules [l]. The reaction, initiated by hydrogen peroxide, provides two oxidising equivalents to the resting haem prosthetic group and yields compound I, an oxy-ferryl-[Fe(IV) = 01-based porphyrin radical cation. Although compounds I and 11, the high oxidation state intermediates formed during the catalytic cycle, have been well described [2], continued efforts to understand structure/function relationships in HRP have been hampered by the lack of a high-resolution structure of a class 111 (higher plant) peroxidase. Despite the wealth of biocheniical data for this archetypal peroxidase it has been necessary to rely
In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.
In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.