Site-directed mutagenesis of pyridoxal kinase from the brain

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Site-directed mutagenesis of pyridoxal kinase from the brain

 

Author: Wong, Ho-yan
Title: Site-directed mutagenesis of pyridoxal kinase from the brain
Year: 2000
Subject: Enzymes -- Analysis
Site-specific mutagenesis
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Applied Biology and Chemical Technology
Pages: vii, 120 leaves : ill. ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1517793
URI: http://theses.lib.polyu.edu.hk/handle/200/2810
Abstract: Site-directed mutagenesis was used to study the structure-function relationships of porcine brain pyridoxal kinase. Three mutants (Y137F, G242A and G244A) of the pyridoxal kinase were successfully constructed and the point mutations were confirmed by DNA sequencing. The mutants and wild-type were overexpressed in E. coli BL21 (DE3) expression system, in which the pyridoxal kinase gene was put under the control of a T7 phage promoter. The expressed proteins were purified using ammonium sulfate fractionation and ion exchange chromatography. Activities of the purified enzymes were measured by using the phenylhydrazine and fluorescence assay. The Y137F mutant was found to lose about 80% of the activity while the activities of the G242A and G244A mutants were found to be totally aborted. Structural and binding properties of the mutants were also analyzed. Results from the guanidinium HCI unfolding study, chymotrypsin digestion, Western blotting and potassium iodide quenching of the mutants and wild-type suggested that the substitution of amino acid residues in our mutants did not cause a dramatic conformational change in the enzyme structure, although changes in the micro-environment was revealed. In addition, the binding of substrate analogs (TNP-ATP and pyridoxaloxime) to the catalytic site of the mutants and wild-type were examined by flurorescence spectroscopy. The results clearly demonstrated that binding ability between the substrates and mutants was adversely affected. Our results also suggested that Tyr137 is located in or around the pyridoxal-binding site of porcine brain pyridoxal kinase. Its involvement may be the formation of hydrogen bond between pyridoxal kinase and pyridoxal. On the other hand, although both Gly242 and Gly244 residues were located in the Gly-rich loop that has been suggested as an unique feature of the ATP-binding site, our results were not favor of a direct critical role of Gly244 in ATP binding. Rather than direct involvement in catalysis, Gly242 and Gly244 were proposed to be involved in maintaining the flexibility of the Gly-rich loop and so indirectly facilitating the substrate binding in pyridoxal kinase.

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