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|標題:||Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis||作者:||Chi, M.C.
|關鍵字:||Bacillus kaustophilus;Leucine aminopeptidase;Computer modeling;Site-directed mutagenesis;Tryptophan emission fluorescence;Circular;dichroism;diolate transition-state;bovine lens;escherichia-coli;active-site;peptide hydrolysis;binding;mechanism;protein;purification;model||Project:||International Journal of Biological Macromolecules||期刊/報告no：:||International Journal of Biological Macromolecules, Volume 43, Issue 5, Page(s) 481-487.||摘要:||
Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants. (C) 2008 Elsevier B.V. All rights reserved.
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