Division of Structural Biology and Bio-informatics, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S.C. Mullick Road, Kolkata, India.
Arch Biochem Biophys. 2011 Nov;515(1-2):21-7. doi: 10.1016/j.abb.2011.08.007. Epub 2011 Aug 27.
The active site architecture of Leishmania major peroxidase (LmP) is very similar with both cytochrome c peroxidase and ascorbate peroxidase. We utilized point mutagenesis to investigate if the conserved proximal methionine residues (Met248 and Met249) in LmP help in controlling catalysis. Steady-state kinetics of methionine mutants shows that ferrocytochrome c oxidation is <2% of wild type levels without affecting the second order rate constant of first phase of Compound I formation, while the activity toward a small molecule substrate, guaiacol or iodide, increases. Our diode array stopped-flow spectral studies show that the porphyrin π-cation radical of Compound I in mutant LmP is more stable than wild type enzyme. These results suggest that the electronegative sulfur atoms of the proximal pocket are critical factors for controlling the location of a stable Compound I radical in heme peroxidases and are important in the oxidation of ferrocytochrome c.
利什曼原虫过氧化物酶（LmP）的活性位点结构与细胞色素 c 过氧化物酶和抗坏血酸过氧化物酶非常相似。我们利用定点突变来研究 LmP 中保守的近端蛋氨酸残基（Met248 和 Met249）是否有助于控制催化。甲硫氨酸突变体的稳态动力学表明，亚铁细胞色素 c 的氧化作用<野生型水平的 2%，而不影响第一阶段复合物 I 形成的二级速率常数，同时对小分子底物愈创木酚或碘化物的活性增加。我们的二极管阵列停流光谱研究表明，突变体 LmP 中的原卟啉 π-阳离子自由基比野生型酶更稳定。这些结果表明，近端口袋的带负电荷的硫原子是控制血红素过氧化物酶中稳定的复合物 I 自由基位置的关键因素，并且对于亚铁细胞色素 c 的氧化很重要。
