2Y6A

Ascorbate Peroxidase R38A mutant

Summary for 2Y6A

• Entry DOI: 10.2210/pdb2y6a/pdb
• Related: 1OAF 1OAG 1V0H 2CL4 2GGN 2GHC 2GHD 2GHE 2GHH 2GHK 2VCF 2VCN 2VCS 2VNX 2VNZ 2VO2 2WD4 2XI6 2XJ6 2Y6B
• Descriptor: ASCORBATE PEROXIDASE, PROTOPORPHYRIN IX CONTAINING FE, SULFATE ION, ... (4 entities in total)
• Functional Keywords: oxidoreductase, heme peroxidase, peroxide scavenger
• Biological source: GLYCINE MAX (SOYBEAN)
• Total number of polymer chains: 1
• Total formula weight: 27678.87

Authors

Metcalfe, C.L.,Efimov, I.,Gumiero, A.,Raven, E.L.,Moody, P.C.E. (deposition date: 2011-01-20, release date: 2011-10-12, Last modification date: 2023-12-20)

Primary citation

Efimov, I.,Badyal, S.K.,Metcalfe, C.L.,Macdonald, I.,Gumiero, A.,Raven, E.L.,Moody, P.C.E.
Proton Delivery to Ferryl Heme in a Heme Peroxidase: Enzymatic Use of the Grotthuss Mechanism.
J.Am.Chem.Soc., 133:15376-, 2011

PubMed Abstract: We test the hypothesized pathway by which protons are passed from the substrate, ascorbate, to the ferryl oxygen in the heme enzyme ascorbate peroxidase (APX). The role of amino acid side chains and bound solvent is demonstrated. We investigated solvent kinetic isotope effects (SKIE) for the wild-type enzyme and several site-directed replacements of the key residues which form the proposed proton path. Kinetic constants for H(2)O(2)-dependent enzyme oxidation to Compound I, k(1), and subsequent reduction of Compound II, k(3), were determined in steady-state assays by variation of both H(2)O(2) and ascorbate concentrations. A high value of the SKIE for wild type APX ((D)k(3) = 4.9) as well as a clear nonlinear dependence on the deuterium composition of the solvent in proton inventory experiments suggest the simultaneous participation of several protons in the transition state for proton transfer. The full SKIE and the proton inventory data were modeled by applying Gross-Butler-Swain-Kresge theory to a proton path inferred from the known structure of APX. The model has been tested by constructing and determining the X-ray structures of the R38K and R38A variants and accounts for their observed SKIEs. This work confirms APX uses two arginine residues in the proton path. Thus, Arg38 and Arg172 have dual roles, both in the formation of the ferryl species and binding of ascorbate respectively and to facilitate proton transfer between the two.

PubMed: 21819069
DOI: 10.1021/JA2007017

Experimental method

X-RAY DIFFRACTION (2 Å)