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Substrate Binding And Catalytic Mechanism In Ascorbate Peroxidase: Evidence For Two Ascorbate Binding Sites.

L. Lad, M. Mewies, E. Raven
Published 2002 · Medicine, Chemistry

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The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.



This paper is referenced by
10.1007/978-3-319-40713-5_6
The Auxin-Nitric Oxide Highway: A Right Direction in Determining the Plant Root System
Natalia Correa-Aragunde (2016)
10.1039/b210426c
Understanding functional diversity and substrate specificity in haem peroxidases: what can we learn from ascorbate peroxidase?
E. Raven (2003)
10.1016/J.BBAGEN.2006.10.001
Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase.
Subhankar Dolai (2007)
10.1038/nbt.2375
Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy
Jeffrey D. Martell (2012)
10.1042/BCJ20170349
Ascorbate protects the diheme enzyme, MauG, against self-inflicted oxidative damage by an unusual antioxidant mechanism.
Zhongxin Ma (2017)
10.1021/acscatal.9b05129
Rewiring the “Push-Pull” Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code
M. Ortmayer (2020)
10.1007/978-3-319-40713-5
Gasotransmitters in Plants
L. Lamattina (2016)
10.1038/s41467-019-10082-7
4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase
Jaime Barros (2019)
10.1016/j.bbapap.2008.03.019
Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).
C. Cooper (2008)
10.1016/j.plaphy.2013.10.009
Tc-cAPX, a cytosolic ascorbate peroxidase of Theobroma cacao L. engaged in the interaction with Moniliophthora perniciosa, the causing agent of witches' broom disease.
L. Camillo (2013)
10.1016/J.ICA.2012.10.031
The kinetics of redox reaction of gold(III) chloride complex ions with l-ascorbic acid
M. Luty-Błocho (2013)
10.1039/c2dt32455e
Probing the conformational mobility of the active site of a heme peroxidase.
A. Gumiero (2013)
10.1038/ncomms13445
Direct visualization of a Fe(IV)–OH intermediate in a heme enzyme
Hanna Kwon (2016)
10.1073/pnas.1618611114
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)
M. Hugo (2017)
Tryptophan oxidation by the heme-containing dioxygenases
E. S. Booth (2016)
Directed evolution of APEX2 for electron microscopy and proximity labeling
Kimberli J Kamer (2014)
10.1074/JBC.M402384200
Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases*
R. Pierattelli (2004)
10.1074/jbc.M602602200
Conformational Mobility in the Active Site of a Heme Peroxidase*
S. K. Badyal (2006)
10.1002/bip.21674
Structural evidence for stabilization of inhibitor binding by a protein cavity in the dehaloperoxidase-hemoglobin from Amphitrite ornata.
Vesna de Serrano (2012)
10.1016/j.bbapap.2008.02.006
Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme.
Rajesh K. Yadav (2008)
10.1007/s11120-006-9100-x
Ascorbate peroxidase–thioredoxin interaction
E. Gelhaye (2006)
10.1039/B309648N
A new framework for understanding substrate binding and functional diversity in haem peroxidases
K. Sharp (2003)
10.1016/j.plantsci.2018.05.013
Primary metabolism changes triggered in soybean leaves by Fusarium tucumaniae infection.
Romina G Rosati (2018)
10.1042/BJ20091406
Euglena gracilis ascorbate peroxidase forms an intramolecular dimeric structure: its unique molecular characterization.
Takahiro Ishikawa (2010)
10.1042/BJ20050311
Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain.
S. Adak (2005)
10.1021/la5012617
Colloidal beading: sonication-induced stringing of selenium particles.
Choon Hwee Bernard Ng (2014)
10.1111/J.1365-313X.2006.02919.X
Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death.
M. D. de Pinto (2006)
10.1074/jbc.M806122200
Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase*
J. Tejero (2008)
10.1104/pp.113.222703
S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells1[OPEN]
M. D. de Pinto (2013)
10.1002/jcc.20446
QM/MM modeling of compound I active species in cytochrome P450, cytochrome C peroxidase, and ascorbate peroxidase
J. Harvey (2006)
Proteome analysis of dissected barley seed tissue during germination and radicle elongation: Heterologous expression of barley limit dextrinase inhibitor
B. Bønsager (2007)
10.1101/2020.08.18.255851
Ascorbate peroxidase neofunctionalization at the origin of APx-R and APx-L: evidences from basal Archaeplastida
F. Lazzarotto (2020)
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