Probing the catalytically essential residues of a recombinant dipeptidyl carboxypeptidase from <Emphasis Type="Italic">Escherichia coli</Emphasis> |
| |
Authors: | Huei-Fen Lo Hsiang-Ling Chen Shao-Yu Yen Ping-Lin Ong Wen-Shiue Chang Chen-Tien Chang |
| |
Institution: | 1.Department of Food Science and Applied Biotechnology,Hungkuang University,Shalu, Taichung,Taiwan;2.Department of Biochemical Science and Technology,National Chiayi University,Chiayi,Taiwan;3.Department of Food and Nutrition,Providence University,Shalu, Taichung,Taiwan |
| |
Abstract: | In studying the structure and function of Escherichia coli dipeptidyl carboxypeptidase (EcDCP), we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme in order to gain insight into the catalytic
mechanism. Comparison of the amino acid sequence of EcDCP with other homologues indicates that the active site of the enzyme exhibits an HEXXH motif, a common feature of zinc metalloenzymes.
The third metal binding ligand, presumed to coordinate directly to the active-site zinc ion in concert with His470 and His474
has been proposed as Glu499. Alterations to these residues completely abolished the catalytic activity against N-benzoyl-l-glycyl-l-histidyl-l-leucine. A significant loss of the enzymatic activity was also observed in F472V and F500V mutant enzymes. Intrinsic tryptophan
fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in all mutant enzymes,
whereas circular dichroism spectra were nearly identical for the tested proteins. Computer modeling suggests that residues
His470, Glu471, His474, Glu499, and Phe500 are essential for EcDCP in maintaining the stable active-site environment. Taken together, these studies contribute to a more comprehensive understanding
of the catalytic mechanism of the enzyme. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|