Bhattacharyya D K, Bandyopadhyay U, Banerjee R K
Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
J Biol Chem. 1993 Oct 25;268(30):22292-8.
Horseradish peroxidase, when incubated with diethyl pyrocarbonate (DEPC), a histidine-specific reagent, shows time-dependent inactivation to oxidize aromatic electron donor, guaiacol. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.67 min-1 M-1. The pH dependence of inactivation shows an inflection point at 6.02, indicating histidine derivatization by DEPC. A difference spectrum of modified versus native enzyme shows a peak at 244 nm for N-carbethoxyhistidine that is diminished by hydroxylamine. Stoichiometric studies indicate that out of 2 histidine residues modified, one is responsible for inactivation. A plot of log reciprocal half-time of inactivation against log DEPC concentration suggests that only 1 histidine is essential. From the computer-stimulated structure of horseradish peroxidase, we tentatively suggest that this critical histidine is most likely distal histidine 42. Binding studies show that this histidine is not involved in guaiacol binding. Modified enzyme forms compound I with H2O2 but not compound II, suggesting a block of electron transfer process. Modified compound I cannot oxidize guaiacol as evidenced by the absence of donor-induced spectral shift from 408 nm, suggesting a block of electron transfer from bound donor to compound I. We suggest that this tentatively identified distal histidine controls aromatic donor oxidation by regulating electron transport without affecting donor binding or compound I formation.
辣根过氧化物酶与组氨酸特异性试剂焦碳酸二乙酯（DEPC）孵育时，氧化芳香族电子供体愈创木酚的活性呈现出时间依赖性失活。失活遵循假一级动力学，二级速率常数为0.67 min⁻¹ M⁻¹。失活的pH依赖性在6.02处出现拐点，表明DEPC对组氨酸进行了衍生化。修饰酶与天然酶的差示光谱显示，N - 乙氧羰基组氨酸在244 nm处有一个峰，该峰可被羟胺减弱。化学计量学研究表明，在2个被修饰的组氨酸残基中，有一个是导致失活的原因。失活的对数倒数半衰期与DEPC浓度的对数作图表明只有1个组氨酸是必需的。根据辣根过氧化物酶的计算机模拟结构，我们初步认为这个关键的组氨酸很可能是远端组氨酸42。结合研究表明，这个组氨酸不参与愈创木酚的结合。修饰后的酶能与H₂O₂形成化合物I，但不能形成化合物II，这表明电子传递过程受阻。修饰后的化合物I不能氧化愈创木酚，从408 nm处没有供体诱导的光谱位移可以证明，这表明从结合的供体到化合物I的电子传递受阻。我们认为，这个初步确定的远端组氨酸通过调节电子传递来控制芳香族供体的氧化，而不影响供体结合或化合物I的形成。
