| Summary: | 碩士 === 國立交通大學 === 生物醫學研究所 === 96 === Abstract
Earlier investigations in our laboratory, a newly defined aminoacylhistidine dipeptidase from Vibrio alginolyticus ATCC 17749 was characterized via the determination of the corresponding gene sequence. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of the peptidase family, which catalyzes the cleavage and release of N-terminal amino acid from a dipeptide molecule like L-carnosine (β-Ala-L-His). The pepD gene from Vibrio alginolyticus encodes a polypeptide of 490 amino acids, which has a sequence that is highly similar to that of dipeptidases from various other species. The researches on bacterial PepD were less known and only investigated genetically and biochemically. Previously, the gene was cloned into the pET-28a(+) expression vector and expressed as a (His)6-PepD fusion protein, which was purified via a Ni-NTA column. The sequence alignment and structure model of PepD indicate that His80, Asp119, Glu150, Asp173 and His461 are metal binding site residues, and Asp82, Glu149, His219, Asn260, Arg369, and Gly435 are putative catalysis and substrate binding residues. Site-directed mutations of Asp82, Glu150 and Arg369 residues of PepD exhibit a decline or loss of activity, suggesting that these residues might be involved in substrate and metal binding, thereby dramatically affecting enzymatic activity. The native Zn2+ ion was removed from PepD using an EDTA chelating solution, which resulted in the loss of hydrolysis activity. However, PepD activity could be restored by adding Mg2+, Mn2+, Co2+, Ni2+, Cu2+ or Cd2+, indicating the functional importance of the metal ion. The functional role of these residues in enzyme catalysis and the effect of metal ions will be discussed in this thesis.
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