INACTIVATION AND DESTRUCTION OF CONSERVED TRP159 OF FE-SUPEROXIDE DISMUTASE FROM PORPHYROMONAS-GINGIVALIS BY HYDROGEN-PEROXIDE

Citation
F. Yamakura et al., INACTIVATION AND DESTRUCTION OF CONSERVED TRP159 OF FE-SUPEROXIDE DISMUTASE FROM PORPHYROMONAS-GINGIVALIS BY HYDROGEN-PEROXIDE, European journal of biochemistry, 253(1), 1998, pp. 49-56
Citations number
46
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0014-2956
Volume
253
Issue
1
Year of publication
1998
Pages
49 - 56
Database
ISI
SICI code
0014-2956(1998)253:1<49:IADOCT>2.0.ZU;2-8
Abstract
The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligat e anaerobe, was purified from Escherichia coli (sodA sodB mutant) harb oring the P. gingivalis SOD-encoding gene. The purified protein contai ned both iron and a small amount of manganese. Iron-and manganese-rcco nstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, resp ectively. These values were similar to the specific activity of the na tive enzyme purified from the recombinant E. coli strain. The Fe-recon stituted 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 af ter 80 min. A concomitant increase in absorbance at 320 nm was observe d with inactivation of the Fe-reconstituted enzyme. Amino acid analysi s of the inactivated Fe-reconstituted enzyme showed a decrease of abou t 0.7 residues of tryptophan/subunit, a value similar to the iron cont ent of the iron-reconstituted enzyme. Three major peptides of the dige sts of the purified SOD with lysylendopeptidase were separated by a re verse-phase HPLC monitoring at 280 nm. One of the peptides, correspond ing to the residues from Gly149 to Lys176, decreased in the HPLC eluen t 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 l ocated midway between the third and fourth metal ligands, Asp157 and H is161, and is conserved in aligned amino acid sequences of all known F e-SODs and Mn-SODs. Based on these results, we propose that the differ ences 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 o f the iron center in the Fe-SODs.