Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase.
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 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). 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-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.
Hill
A P
AP
Department of Chemistry, University of Leicester, England, UK.
Modi
S
S
Sutcliffe
M J
MJ
Turner
D D
DD
Gilfoyle
D J
DJ
Smith
A T
AT
Tam
B M
BM
Lloyd
E
E
D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004789:Enzyme Activation; D006418:Heme / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D010659:Phenylhydrazines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D010659:Phenylhydrazines; C030299:phenylhydrazine; D006418:Heme; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid