Yamakura F, Rardin R L, Petsko G A, Ringe D, Hiraoka B Y, Nakayama K, Fujimura T, Taka H, Murayama K
Department of Chemistry, Juntendo University School of Medicine, Inba, Chiba, Japan.
Eur J Biochem. 1998 Apr 1;253(1):49-56. doi: 10.1046/j.1432-1327.1998.2530049.x.
The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5% of its original activity after a 15 min incubation at 25 degrees C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20% of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properties of the iron center in the Fe-SODs.
牙龈卟啉单胞菌（一种专性厌氧菌）的超氧化物歧化酶（SOD）是从携带牙龈卟啉单胞菌SOD编码基因的大肠杆菌（sodA sodB突变体）中纯化得到的。纯化后的蛋白质含有铁和少量锰。仅含有这两种金属之一的铁重构SOD和锰重构SOD的比活性分别为1000和1200 U/mg/金属/亚基摩尔。这些值与从重组大肠杆菌菌株中纯化得到的天然酶的比活性相似。在25℃、pH 7.8条件下孵育15分钟后，10 mM过氧化氢可使铁重构酶失活至其原始活性的约5%，而锰重构酶在80分钟后未显示失活。随着铁重构酶的失活，在320 nm处观察到吸光度同时增加。对失活的铁重构酶进行氨基酸分析表明，色氨酸/亚基残基减少了约0.7个，该值与铁重构酶的铁含量相似。用赖氨酰内肽酶消化纯化后的SOD得到的三种主要肽段，通过在280 nm处进行反相HPLC监测进行分离。其中一种肽段，对应于从Gly149到Lys176的残基，在H2O2失活的SOD的HPLC洗脱液中减少至天然SOD测量量的20%。由于该肽段仅含有一个色氨酸残基，因此得出结论，分解的色氨酸残基是Trp159，它位于第三个和第四个金属配体Asp157和His161之间的中间位置，并且在所有已知的铁SOD和锰SOD的比对氨基酸序列中保守。基于这些结果，我们提出，铁SOD和锰SOD对过氧化氢敏感性的差异可能是由铁SOD和锰SOD活性位点金属的身份差异以及铁SOD中铁中心性质的调整引起的。
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