Substrate Binding and Catalytic Mechanism in Ascorbate Peroxidase: Evidence for Two Ascorbate Binding Sites

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Abstract
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 × 107 M-1 s-1) of Compound I and reduction (k2 = 5.2 ± 0.3 × 106 M-1 s-1) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (λmax (nm) = 409, 530, 569, 655) are consistent with a porphyrin π-cation radical. Reduction of Compound II by l-ascorbate is rate-limiting: at low substrate concentration (0−500 μM), kinetic traces were monophasic but above ∼500 μM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 μM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k3-slow = 3.1 ± 0.1 × 103 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 (k3 = 0.9 ± 0.1 × 103 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.