Study of human glutaminyl cyclase mutants and mutant E201Q/substrate complex structure on the influence of enzyme catalytic mechanism
Date Issued
2005
Date
2005
Author(s)
Liu, Yi-Liang
DOI
en-US
Abstract
Glutaminyl cyclase (QC) is a critical enzyme that is responsible for the conversion of N-terminal glutamine into pyroglutamic acid, which is an important event during protein synthesis. Many hormones, such as thyrotropin-releasing hormone and neurotensin, need QCs for modifying their N-terminal glutamine and subsequently become an active form. In the previous studies, QCs from various species, such as porcine, bovine, papaya or human have been cloned, expressed and characterized. However, the exact catalytic process still remained ambiguous due to the lack of ingormation on QC structure.
The human QC has been cloned and expressed in large scale and purified successfully by Kai-Fa Huang in our group. He also determined the first QC and QC/inhibitors structures by x-ray crystallography (2005 in press). Enzyme catalytic mechanism was proposed preliminarily based on the structural alignment and the solved structures. In order to confirm the catalytic mechanism proposed by Huang, some key residues, like E201, D248, D305, Q304, H307, H319, F325 and W329, which are likely to involve in the catalytic mechanisms or play vital roles in the substrate recognition, have been chosen for site-directed mutagenesis. The catalytic ability of wild-typed human QC was assessed and characterized by high performance liquid chromatography. Coupled enzyme assay was used to evaluate the activities of the mutants. Among them, E201Q, which dramatically lost its catalytic activity, was subjected to further investigation. The E201Q/glutamine-t-butyl ester complex structure was determined. The structural information of substrate binding is supportive to the previous suggestion on enzyme catalysis. Combined with results from the biological activity analysis of human QC mutants, the proposed mechanism could be more solid and reliable.
Subjects
麩醯胺環化酵素
催化機轉
X-光結晶學
定點突變
glutaminyl cyclase
catalytic mechanism
X-ray crystallography
site-directed mutagenesis
Type
other
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