Author: Fung, Long-yan
Title: Structure and function relationship of porcine pyridoxal kinase
Degree: M.Phil.
Year: 1999
Subject: Enzymes -- Analysis
Proteins -- Analysis
Hong Kong Polytechnic University -- Dissertations
Department: Department of Applied Biology and Chemical Technology
Pages: [19], 152 leaves : ill. ; 30 cm
Language: English
Abstract: Recombinant truncated mutants of pyridoxal kinase lacking a series of amino acid residues at the N-terminal domain have been constructed. Deletion of 15 amino acid residues does not inactivate the kinase, whereas deletion of 16, 17, 18, and 21 or more amino acid residues, pertaining to the highly conserved sequence, RVLSIQHV, abolishes the catalytic function of the expressed enzyme. All species of recombinant pyridoxal kinase were purified using a method that a chain of histidine residues were tagged at the N-terminal of the protein. Wildtype, Δ15 and Δ16 variants of pyridoxal kinase were puritied to homogeneity by two chromatographic steps including metal-chelating and DEAE ion exchange chromatographies. Purified enzymes were characterized by using circular dichroism (CD) and fluorescence spectroscopy with respect to the folding pattern of the protein. Results indicate that deletion of 16 amino acid residues initiates a misfolding pattern for the protein leading to subsequent loss of their ability to bind an ATP analogue, Trinitrophenyl-ATP (TNP-ATP), in vitro. Thc stability of wild-type and Δ15 mutant in the presence of guandinium hydrochloride (GdnHCl) was also examined using fluorescence spectroscopy. Results have shown that the unfolding process of the wild-type protein proceeds through a concerted reaction, whereas the denaturation process of the mutants (Δ15 and Δ16) endowed with and without catalytic activity shows a biphasic pattern of unfolding in GdnHCI. It is concluded that deletion mutants of pyridoxal kinase are not only less stable than the wild-type species, but they also follow different folding pathways.
Rights: All rights reserved
Access: open access

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