Lad Latesh, Mewies Martin, Raven Emma Lloyd
Department of Chemistry, University of Leicester, UK.
Biochemistry. 2002 Nov 19;41(46):13774-81. doi: 10.1021/bi0261591.
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.
利用预稳态和稳态动力学技术，研究了重组大豆胞质抗坏血酸过氧化物酶（rsAPX）及其一个衍生物的催化机制。在该衍生物中，靠近底物（L-抗坏血酸）结合位点的一个半胱氨酸残基（Cys32）已被修饰，以阻止抗坏血酸的结合[曼德尔曼，D.，贾马尔，J.，和普洛斯，T. L.（1998年）《生物化学》37卷，17610 - 17617页]。化合物I的形成（k1 = 3.3±0.1×10⁷ M⁻¹ s⁻¹）以及底物对化合物I的还原（k₂ = 5.2±0.3×10⁶ M⁻¹ s⁻¹）都很快。rsAPX的化合物I的波长最大值（λmax（纳米）= 409、530、569、655）与卟啉π-阳离子自由基一致。L-抗坏血酸对化合物II的还原是限速步骤：在低底物浓度（0 - 500微摩尔）下，动力学曲线是单相的，但在约500微摩尔以上是双相的。快速相的观测速率常数与从500微摩尔以下观测到的（单相）依赖性中提取的观测速率常数重叠，并显示出饱和动力学；慢相的速率常数与底物浓度呈线性相关（k(3 - slow)）= 3.1±0.1×10³ M⁻¹ s⁻¹）。对于Cys32修饰的rsAPX，L-抗坏血酸还原化合物II的动力学瞬变在所有底物浓度下都是单相的，二级速率常数（k₃ = 0.9±0.1×¹⁰³ M⁻¹ s⁻¹）与从未修饰的rsAPX的化合物II还原慢相中获得的速率常数相似。rsAPX对L-抗坏血酸的稳态氧化显示出对底物浓度的S形依赖性，并且使用希尔方程对数据进行了令人满意的合理化处理；Cys32修饰的rsAPX对L-抗坏血酸的氧化没有显示出S形行为的证据。这些数据与APX中存在两个对抗坏血酸具有动力学活性的结合位点一致。
