Miller V P, Goodin D B, Friedman A E, Hartmann C, Ortiz de Montellano P R
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
J Biol Chem. 1995 Aug 4;270(31):18413-9. doi: 10.1074/jbc.270.31.18413.
A gene coding for the F172Y mutant of horseradish peroxidase isozyme C (HRP) has been constructed and expressed in both Spodoptera frugiperda (SF-9) and Trichoplusia ni egg cell homogenate (HighFive) cells. Homology modeling with respect to three peroxidases for which crystal structures are available places Phe172 on the proximal side of the heme in the vicinity of porphyrin pyrrole ring C. The pH optimum and spectroscopic properties of the F172Y mutant are essentially identical to those of wild type HRP. Vmax values show that the mutant protein retains most of the guaiacol oxidizing activity. Stopped flow studies indicate that Compound I is formed with H2O2 at the same rate (kappa 1 = 1.6 x 10(7) M-1 s-1) at both pH 6.0 and 8.0 as it is with the wild type enzyme. This Compound I species decays rapidly at a rate kappa 2 = 1.01 s-1, pH 7.0, to a second two-electron oxidized species that retains the ferryl (FeIV = O) absorption. EPR studies establish that a ferryl porphyrin radical cation is present in the initial Compound I, but electron transfer from the protein results in formation of a second Compound I species with an unpaired electron on the protein (presumably on Tyr172). The presence or absence of oxidizable amino acids adjacent to the heme is thus a key determinant of whether the second oxidation equivalent in Compound I is found as a porphyrin or protein radical cation.
已构建了编码辣根过氧化物酶同工酶C（HRP）F172Y突变体的基因，并在草地贪夜蛾（SF-9）细胞和粉纹夜蛾卵细胞匀浆（HighFive）细胞中进行了表达。对三种已知晶体结构的过氧化物酶进行同源建模，结果表明苯丙氨酸172位于血红素近端，靠近卟啉吡咯环C。F172Y突变体的最适pH值和光谱性质与野生型HRP基本相同。Vmax值表明突变蛋白保留了大部分愈创木酚氧化活性。停流研究表明，在pH 6.0和8.0时，突变体与野生型酶一样，与H2O2以相同速率（κ1 = 1.6 x 10(7) M-1 s-1）形成化合物I。该化合物I物种在pH 7.0时以κ2 = 1.01 s-1的速率迅速衰减为第二种双电子氧化物种，该物种保留了铁（IV）=氧的吸收。电子顺磁共振研究表明，初始化合物I中存在铁卟啉自由基阳离子，但蛋白质的电子转移导致形成第二种化合物I物种，其蛋白质上存在未成对电子（可能在酪氨酸172上）。因此，血红素附近是否存在可氧化氨基酸是决定化合物I中第二个氧化当量是卟啉自由基阳离子还是蛋白质自由基阳离子的关键因素。
