Borders C L, Bjerrum M J, Schirmer M A, Oliver S G
Department of Chemistry, The College of Wooster, Ohio 44691, USA.
Biochemistry. 1998 Aug 11;37(32):11323-31. doi: 10.1021/bi980167t.
All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role [Chan et al., Arch. Biochem. Biophys. 279, 195-201 (1990)]. In Saccharomyces cerevisiae MnSOD, the active-site arginine is replaced by a lysine. The S. cerevisiae MnSOD gene has been cloned and expressed in E. coli, and H30A and K170R site-specific mutants have been prepared. The purified recombinant native (RN) and mutant enzymes were compared to one another and to the native enzyme purified from S. cerevisiae (SC) in terms of activity, temperature stability, and sensitivity to 2,4,6-trinitrobenzenesulfonate (TNBS) and phenylglyoxal (PG). All enzymes had high specific activities (SC = 5000, RN = 5600, H30A = 4500, K170R = 4600) (U/mg, using the pyrogallol assay). SC, RN, and H30A were very stable at 75 degreesC (pH 8.0), with half-lives of 4.7, 2.8, and 2.7 h, respectively, while K170R had a much greater temperature lability, with a half-life of 0.36 h under these conditions. TNBS (0.5 mM, pH 9.0, 25 degreesC) rapidly inactivated SC, RN, and H30A, with half-lives of 3. 5, 5.1, and 5.5 min, respectively, but only slowly inactivated K170R, with a half-life of 101 min. PG (20 mM, pH 9.0, 25 degreesC) caused very slow inactivation of SC, RN, and H30A by biphasic kinetics, and each enzyme retained >/=25% activity after 3 h of modification. K170R, on the other hand, was completely inactivated by PG under these conditions by first-order kinetics, with a half-life of 7.0 min. The data suggest that His-30, a residue highly conserved in the active-site channel of MnSODs and FeSODs, does not play a crucial role in catalysis or stability. In addition, Lys-170, a residue that is almost always arginine in the numerous other MnSODs and FeSODs sequenced to date, can be replaced by arginine with no loss of catalytic activity, but K170R is less stable and Arg-170 in this mutant is more exposed than the corresponding arginine in other SODs. RN and SC showed some surprising differences. Thus, while the specific activities of RN and SC are very similar, SC is more stable to inactivation at 75 degreesC, and less susceptible to inactivation by phenylglyoxal, than RN. These data suggest that there may be slight differences in the tertiary structures of SC, the native enzyme expressed in S. cerevisiae, and RN, the recombinant native enzyme expressed in E. coli.
所有已知含锰超氧化物歧化酶（MnSODs）在活性位点通道中都有一个高度保守的组氨酸（大肠杆菌铁超氧化物歧化酶中的His-30），并且几乎所有酶都有一个活性位点精氨酸（Arg-170），有人提出该精氨酸具有结构和功能双重作用[Chan等人，《生物化学与生物物理学报》279, 195 - 201 (1990)]。在酿酒酵母MnSOD中，活性位点精氨酸被赖氨酸取代。酿酒酵母MnSOD基因已被克隆并在大肠杆菌中表达，且已制备了H30A和K170R位点特异性突变体。对纯化的重组天然（RN）酶和突变酶，以及从酿酒酵母（SC）中纯化的天然酶，在活性、温度稳定性以及对2,4,6 - 三硝基苯磺酸盐（TNBS）和苯乙二醛（PG）的敏感性方面进行了比较。所有酶都具有高比活性（SC = 5000，RN = 5600，H30A = 4500，K170R = 4600）（U/mg，使用连苯三酚测定法）。SC、RN和H30A在75℃（pH 8.0）时非常稳定，半衰期分别为4.7、2.8和2.7小时，而K170R在温度稳定性方面要差得多，在这些条件下半衰期为0.36小时。TNBS（0.5 mM，pH 9.0，25℃）能快速使SC、RN和H30A失活，半衰期分别为3.5、5.1和5.5分钟，但只能缓慢使K170R失活，半衰期为101分钟。PG（20 mM，pH 9.0，25℃）通过双相动力学使SC、RN和H30A非常缓慢地失活，且在修饰3小时后每种酶仍保留≥25%的活性。另一方面，在这些条件下K170R被PG通过一级动力学完全失活，半衰期为7.0分钟。数据表明，His-30这个在MnSODs和FeSODs活性位点通道中高度保守的残基，在催化或稳定性方面不起关键作用。此外，Lys-170这个在迄今为止测序的众多其他MnSODs和FeSODs中几乎总是精氨酸的残基，可以被精氨酸取代而不损失催化活性，但K170R稳定性较差，且该突变体中的Arg-170比其他超氧化物歧化酶中的相应精氨酸更暴露。RN和SC表现出一些惊人的差异。因此，虽然RN和SC的比活性非常相似，但SC在75℃时对失活更稳定，并且比RN更不易被苯乙二醛失活。这些数据表明，在酿酒酵母中表达的天然酶SC和在大肠杆菌中表达的重组天然酶RN的三级结构可能存在细微差异。
