Vitello L B, Erman J E, Mauro J M, Kraut J
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Biochim Biophys Acta. 1990 Mar 29;1038(1):90-7. doi: 10.1016/0167-4838(90)90015-8.
The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.
大肠杆菌产生的细胞色素 c 过氧化物酶（CcP(MI)）与过氧化氢之间的双分子反应，在中性 pH 区域内与天然酵母细胞色素 c 过氧化物酶（CcP）和过氧化氢的反应相同。两种酶与过氧化氢反应的双分子速率常数均与 pH 无关，为 46 μM⁻¹·s⁻¹。第二种突变酶，即大肠杆菌产生的在 191 位带有苯丙氨酸的细胞色素 c 过氧化物酶突变体（CcP(MI, F191)），与过氧化氢反应的双分子速率常数与 pH 无关，为 65 μM⁻¹·s⁻¹，比 CcP 或 CcP(MI)大 40%。与用过氧化氢氧化 CcP 或 CcP(MI)时形成的氧合铁卟啉π-阳离子自由基中间体不同，CcP(MI, F191)的初始过氧化物氧化产物是氧合铁卟啉π-阳离子自由基中间体。在含 KNO₃ 的缓冲液中，这三种酶与过氧化氢的反应都依赖于 pH。这些反应受一个可电离基团的影响，在这三种酶中其表观 pKa 为 5.4。当可电离基团未质子化时，这些酶与过氧化氢发生反应。通过过氧化氢滴定和光谱分析评估，与 CcP 相比，CcP(MI)和 CcP(MI, F191)的 pH 稳定性区域略小。结构稳定性的改变必定归因于 CcP 和 CcP(MI)在 -2、-1、53 和 152 位的一级序列差异。
