Chi Meng-Chun, Ong Ping-Lin, Hsu Wen-Hwei, Chen Yan-Hung, Huang Hsien-Bin, Lin Long-Liu
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi 621, Taiwan.
Int J Biol Macromol. 2008 Dec;43(5):481-7. doi: 10.1016/j.ijbiomac.2008.09.009. Epub 2008 Sep 19.
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.
