L-ascorbate peroxidase

 

L-ascorbate peroxidase (AP) is a heme peroxidase. This group of enzyme catalyse the reduction of hydrogen peroxide (H2O2) to water and the concurrent oxidisation of a range of substrates, in this case ascorbic acid.

Ascorbic acid is a strong antioxidant that is effective in scavenging superoxide (O2-'), hydroxyl (OH') radicals and singlet oxygen. It can also remove H2O2 in the following reaction.

AP is the main enzyme responsible for hydrogen peroxide removal in the chloroplasts and cytosol of higher plants.

 

Reference Protein and Structure

Sequence
P48534 UniProt (1.11.1.11) IPR002207 (Sequence Homologues) (PDB Homologues)
Biological species
Pisum sativum (pea) Uniprot
PDB
1apx - CRYSTAL STRUCTURE OF RECOMBINANT ASCORBATE PEROXIDASE (2.2 Å) PDBe PDBsum 1apx
Catalytic CATH Domains
1.10.520.10 CATHdb 1.10.420.10 CATHdb (see all for 1apx)
Cofactors
Heme b (1), L-ascorbate (1)
Click To Show Structure

Enzyme Reaction (EC:1.11.1.11)

L-ascorbate
CHEBI:38290ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
→
L-dehydroascorbate
CHEBI:58539ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: L-ascorbic acid peroxidase, L-ascorbic acid-specific peroxidase, Ascorbate peroxidase, Ascorbic acid peroxidase,

Enzyme Mechanism

Introduction

The mechanism can be broken down into four steps:

  1. Fe(III) + H2O2 = Compound I (Fe(IV) radical) + H2O. This first step is catalysed by residues on the distal side of the heme group. H2O2 enters the active site and binds to the heme Fe, His 42, and Arg 38. The oxygen of H2O2 that is bound to the Fe donates a proton to the NE of the histidine. The histidine then in turn donates the proton to the other H2O2 oxygen bound to the arginine. This leaves water bound to arginine and the oxidised Compound I. This step is common to all heme peroxidases.
  2. Compound I (Fe(IV) radical) + L-ascorbate = Compound II (Fe(IV)) + monodehydro-L-ascorbate radical
  3. Compound II (Fe(IV)) + L-ascorbate = Peroxidase (Fe(III)) + monodehydro-L-ascorbate radical
  4. 2 monodehydro-L-ascorbate radicals = L-ascorbate + L-dehydroascorbate.

Catalytic Residues Roles

UniProt PDB* (1apx)
Arg38 Arg38(37)A Transition state stabiliser. transition state stabiliser
His42 His42(41)A General acid/base. proton shuttle (general acid/base)
His163 His163(162)A Axial ligand to the heme b cofactor. activator, metal ligand
Trp179 Trp179(178)A Radical stabiliser. There is some suggestion that the Compound I radical may spend some time on this residue. radical stabiliser
Asp208 Asp208(207)A Hydrogen bonds to His163 (axial heme ligand) and Trp179, helping to position and activate them. activator
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Raven EL et al. (2004), Biochem Soc Symp, 71, 27-38. Defining substrate specificity and catalytic mechanism in ascorbate peroxidase. DOI:10.1042/bss0710027.
  2. Adak S et al. (2013), Antioxid Redox Signal, 19, 746-754. Ascorbate Peroxidase Acts As a Novel Determiner of Redox Homeostasis inLeishmania. DOI:10.1089/ars.2012.4745. PMID:22703594.
  3. Sharp KH et al. (2003), Nat Struct Biol, 10, 303-307. Crystal structure of the ascorbate peroxidase–ascorbate complex. DOI:10.1038/nsb913. PMID:12640445.
  4. Lad L et al. (2002), Eur J Biochem, 269, 3182-3192. Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants. PMID:12084058.
  5. Lad L et al. (2002), Biochemistry, 41, 13774-13781. Substrate Binding and Catalytic Mechanism in Ascorbate Peroxidase:  Evidence for Two Ascorbate Binding Sites†. DOI:10.1021/bi0261591.
  6. Hiner ANP et al. (2001), Eur J Biochem, 268, 3091-3098. Detection of a tryptophan radical in the reaction of ascorbate peroxidase with hydrogen peroxide. DOI:10.1046/j.1432-1327.2001.02208.x.
  7. Patterson WR et al. (1995), Biochemistry, 34, 4331-4341. Crystal structure of recombinant pea cytosolic ascorbate peroxidase. DOI:10.1021/bi00013a023. PMID:7703247.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg38(37)A transition state stabiliser
His42(41)A proton shuttle (general acid/base)
His163(162)A metal ligand, activator
Trp179(178)A radical stabiliser
Asp208(207)A activator

Chemical Components

Step 1.

Contributors

Stuart Lucas, Craig Porter, Gemma L. Holliday