O'Neill P, Davies S, Fielden E M, Calabrese L, Capo C, Marmocchi F, Natoli G, Rotilio G
M.R.C. Radiobiology Unit, Chilton, Didcot, Oxon, U.K.
Biochem J. 1988 Apr 1;251(1):41-6. doi: 10.1042/bj2510041.
The CuZn superoxide dismutases (SODs) from ox, sheep, pig and yeast were investigated by pulse radiolysis in order to evaluate the role of electrostatic interactions between O2.- and SOD proteins in the mechanism of action of the SOD enzymes. The protein net charge in this series varies, as evaluated by the protein pI values spanning over a large range of pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox) and 4.6 (yeast). The amino acid sequences are largely conserved, with the three mammalian proteins being highly homologous and the yeast protein having some distinct variations in the region surrounding the active site. At pH 8.0 the activities of the SODs from various sources are similar, though the minor differences observed suggest that in the highly homologous mammalian series the most acidic protein is the most enzymically efficient one. The pH-dependences of the various activities in the pH range 7-12 are similar, and the related curves are best fitted by two pK values, which are approx. 9.2 and 11.0 for the mammalian enzymes and 9.1 and 11.4 for the yeast enzyme. The activities of the proteins at I 0.1 are decreased by approx. 20% when compared with the activity at I 0.02 at pH 8.5, whereas at pH above 10 the pH-dependence of the activity approaches that determined at I 0.02 and at pH 11.9 the activity is essentially independent of ionic strength. The dependence upon ionic strength also depends on the salt used, with perchlorate being more effective than phosphate or borate or Mops and still effective at pH above 10.5, where the effect of other salts becomes negligible. The dual and concerted dependence of the activities of different SODs on pH and salt concentration is explained with the encounter of O2.- with the active-site copper being governed by the protonation of two positively charged groups in the vicinity of the active site. The gradient between these localized charges and the rest of the protein may explain the different activities of the mammalian proteins at lower pH. On the basis of the sequence variation of the SODs examined it is not possible to definitely identify these groups. Likely candidates are conserved basic amino acid side chains in the vicinity (less than or equal to 1.2 nm) of the active site, i.e. Lys-134 and Arg-141, but co-ordination of OH- in the first copper co-ordination sphere may be an additional factor accounting for the higher pK.(ABSTRACT TRUNCATED AT 400 WORDS)
通过脉冲辐解研究了来自牛、羊、猪和酵母的铜锌超氧化物歧化酶（SOD），以评估超氧阴离子（O₂⁻）与SOD蛋白之间的静电相互作用在SOD酶作用机制中的作用。根据蛋白质的pI值评估，该系列中的蛋白质净电荷有所不同，其pI值跨越很大的pH范围：8.0（羊）、6.5（猪）、5.2（牛）和4.6（酵母）。氨基酸序列在很大程度上是保守的，三种哺乳动物蛋白质高度同源，而酵母蛋白质在活性位点周围区域有一些明显的差异。在pH 8.0时，各种来源的SOD活性相似，尽管观察到的微小差异表明，在高度同源的哺乳动物系列中，酸性最强的蛋白质酶活性最高。在pH 7 - 12范围内，各种活性的pH依赖性相似，相关曲线用两个pK值拟合最佳，哺乳动物酶的pK值约为9.2和11.0，酵母酶的pK值约为9.1和11.4。在pH 8.5时，蛋白质在离子强度I 0.1时的活性与在I 0.02时相比降低了约20%，而在pH高于10时，活性的pH依赖性接近在I 0.02时测定的值，在pH 11.9时，活性基本上与离子强度无关。对离子强度的依赖性还取决于所使用的盐，高氯酸盐比磷酸盐、硼酸盐或Mops更有效，在pH高于10.5时仍然有效，此时其他盐的影响可以忽略不计。不同SOD活性对pH和盐浓度的双重协同依赖性可以解释为，O₂⁻与活性位点铜的相遇受活性位点附近两个带正电荷基团的质子化控制。这些局部电荷与蛋白质其余部分之间的梯度可以解释哺乳动物蛋白质在较低pH下的不同活性。根据所研究的SOD的序列变异，无法明确识别这些基团。可能的候选者是活性位点附近（小于或等于1.2 nm）保守的碱性氨基酸侧链，即Lys - 134和Arg - 141，但第一个铜配位球中OH⁻的配位可能是导致较高pK的另一个因素。（摘要截断于400字）
