Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II
Tóm tắt
Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k cat/K m. These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.
Tài liệu tham khảo
citation_journal_title=Mol Immunol; citation_title=The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides; citation_author=AL Goldberg, P Cascio, T Saric, KL Rock; citation_volume=39; citation_publication_date=2002; citation_pages=147-164; citation_doi=10.1016/S0161-5890(02)00098-6; citation_id=CR1
citation_journal_title=Antonie van Leewenhock; citation_title=Histidines 345 and 378 of Bacillus stearothermophilus leucine aminopeptidase II are essential for the catalytic activity of the enzyme; citation_author=GY Hwang, LY Kuo, MR Tsai, SL Yang, LL Lin; citation_volume=87; citation_publication_date=2005; citation_pages=355-359; citation_doi=10.1007/s10482-004-5777-z; citation_id=CR2
citation_journal_title=Curr Microbiol; citation_title=Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus
; citation_author=LY Kuo, GY Hwang, YJ Lai, SL Yang, LL Lin; citation_volume=47; citation_publication_date=2003; citation_pages=40-45; citation_doi=10.1007/s00284-002-3950-z; citation_id=CR3
citation_journal_title=Protein J; citation_title=Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme; citation_author=LY Kuo, GY Hwang, SL Yang, YW Hua, W Chen, LL Lin; citation_volume=23; citation_publication_date=2004; citation_pages=295-302; citation_doi=10.1023/B:JOPC.0000027854.56051.e4; citation_id=CR4
citation_journal_title=Nature (London); citation_title=Cleavage of structural proteins during the assembly of the head of bacteriophage T4; citation_author=UK Laemmli; citation_volume=227; citation_publication_date=1970; citation_pages=680-685; citation_doi=10.1038/227680a0; citation_id=CR5
citation_journal_title=Chem Rev; citation_title=Metalloaminopeptidases: common functional themes in disparate structural surroundings; citation_author=WT Lowther, BW Matthews; citation_volume=102; citation_publication_date=2002; citation_pages=4581-4607; citation_doi=10.1021/cr0101757; citation_id=CR6
citation_journal_title=J Biol Chem; citation_title=
Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan; citation_author=SG Odintsov, I Sabala, G Bourenkov, V Rybin, M Bochtler; citation_volume=280; citation_publication_date=2005; citation_pages=27792-27799; citation_doi=10.1074/jbc.M502023200; citation_id=CR7
citation_journal_title=Proc Natl Acad Sci USA; citation_title=Leucine aminopeptidase: an inducible component of the defense response in Lycopersion esculentum (tomato); citation_author=V Pautot, FM Holzer, B Reisch, LL Walling; citation_volume=90; citation_publication_date=1993; citation_pages=9906-9910; citation_doi=10.1073/pnas.90.21.9906; citation_id=CR8
citation_journal_title=Anal Biochem; citation_title=Spectroscopic methods for analysis of protein secondary structure; citation_author=JT Pelton, LR Mclean; citation_volume=277; citation_publication_date=2000; citation_pages=167-176; citation_doi=10.1006/abio.1999.4320; citation_id=CR9
citation_journal_title=Biochemistry; citation_title=Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme; citation_author=SL Roderick, BW Matthews; citation_volume=32; citation_publication_date=1993; citation_pages=3907-3912; citation_doi=10.1021/bi00066a009; citation_id=CR10
citation_journal_title=Biochim Biophys Acta; citation_title=Aminopeptidase II from Bacillus stearothermophilus
; citation_author=E Stoll, HG Weder, H Zuber; citation_volume=438; citation_publication_date=1976; citation_pages=211-220; citation_id=CR11
citation_journal_title=Peptides; citation_title=Nociceptin/orphanin FQ metabolism and bioactive metabolites; citation_author=L Terenius, J Sandin, T Sakurada; citation_volume=21; citation_publication_date=2000; citation_pages=919-922; citation_doi=10.1016/S0196-9781(00)00228-X; citation_id=CR12
citation_journal_title=Proc Natl Acad Sci USA; citation_title=Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli
; citation_author=MCJ Wilce, CS Bond, NE Dixon, HC Freeman, JM Guss, PE Lilly, JA Wilce; citation_volume=95; citation_publication_date=1998; citation_pages=3472-3477; citation_doi=10.1073/pnas.95.7.3472; citation_id=CR13