CHEMICAL, SPECTROSCOPIC AND STRUCTURAL INVESTIGATION OF THE SUBSTRATE-BINDING SITE IN ASCORBATE PEROXIDASE

The interaction of recombinant ascorbate peroxidase (APX) with its phy siological substrate, ascorbate, has been studied by electronic and NM R spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is cons istent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant, K-d, of 11.6 +/- 0.4 mu M (pH 7.002, m u = 0.10 M, 25.0 degrees C). Individual distances between the non-exch angeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate th at the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction o f ferric APX with the suicide substrate phenylhydrazine yields predomi nantly (60%) a covalent haem adduct which is modified at the C20 carbo n, indicating that substrate binding and oxidation is close to the exp osed C20 position of the haem, as observed for other classical peroxid ases. Molecular-modelling studies, using the NNM-derived distance rest raints in conjunction with the crystal structure of the enzyme [Patter son, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are con sistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III p eroxidases) is implicated.