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Title: Structure and function relationship of enzyme : pyridoxal kinase and trichosanthin
Authors: Lau, Chi-kong
Keywords: Enzymes -- Analysis
Proteins -- Analysis
Vitamin B6
Hong Kong Polytechnic University -- Dissertations
Issue Date: 1999
Publisher: The Hong Kong Polytechnic University
Abstract: Pyridoxal kinase was purified to homogeneity from porcine brain. The purified protein was digested separately with trypsin, chymotrypsin, endo-proteinase Lys-C and endo-proteinase Glu-C and resulting peptides purified by using reverse phase HPLC and SDS-PAGE. Purified peptides were then subjected to ammo acid sequencing and the primary structure of pyridoxal kinase was elucidated. The amino acid sequence of pyridoxal kinase was further analyzed with respect 10 its significance using secondary structure prediction, sequence similarity search and multiple sequence alignment. The boundary of the ATP-binding domain in pyridoxal kinase was identified by amino acid sequencing. The identified domain sequence was then folded using the program THREADER 2. Results have shown that NAD-dependent formate dehydrogenase (2nac) and the ATP-binding domain share similar folding patterns. Using the structure of NAD-dependent formate dehydrogenase as a template, a model was constructed for the ATP-binding domain of pyridoxal kinase using the molecular modeling software, INSIGHT II. The resulting model was further analyzed for its validity by comparison with previous experimental data. Docking of the ATP molecule and its analogue (AP₄PL) to the respective domain has also been undertaken in order to identify the binding site of the domain. The unfolding mechanism of trichosanthin was studied. It was monitored by using fluorescence spectroscopy and circular dichroism in the presence of different concentrations of guanidinium chloride (GdnHCI). Results have shown that trichosanthin is irreversibly denatured by 6 M GdnHCI and intermediates between the unfolded and native structure of the protein can be formed. After unfolding, the denatured trichosanthin was reconstituted in the presence of the ehaperone protein. GroEL. It was found that unfolded trichosanthin could be recognized and reactivated by GroEL in the presence of Mg-ATP.
Description: xvii, 274 leaves : ill. (some col.) ; 30 cm
PolyU Library Call No.: [THS] LG51 .H577P ABCT 1999 Lau
Rights: All rights reserved.
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