Spectroscopic characterization of recombinant pyridoxal kinase mutants

Pao Yue-kong Library Electronic Theses Database

Spectroscopic characterization of recombinant pyridoxal kinase mutants

 

Author: Ng, Kang-to Michael
Title: Spectroscopic characterization of recombinant pyridoxal kinase mutants
Degree: M.Phil.
Year: 2003
Subject: Vitamin B6
Enzymes -- Analysis
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Applied Biology and Chemical Technology
Pages: xiii, 248 leaves : ill. ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1697426
URI: http://theses.lib.polyu.edu.hk/handle/200/4077
Abstract: This project is divided into two parts; the first part is the overexpression and biochemical characterization of recombinant porcine and human pyridoxal kinase. The second part is to investigate the structure-function relationships of porcine pyridoxal kinase by studying the site-directed mutants. In the first part of the project, the recombinant porcine pyridoxal kinase (PPK) and His-tagged human pyridoxal kinase (His-HPK) were overexpressed in E. coli and B. subtilis, respectively. They were purified to homogeneity (18-fold for PPK and 57.5-fold for His-HPK). Both purified enzymes exhibited a molecular mass of about 35 kDa when examined by 12% SDS-PAGE. The results of kinetic analysis showed that the Km values for pyridoxal were 506 +- 49 and 883 +- 69 uM for PPK and His-HPK, respectively, whereas, Km values for ATP were 48 +- 4 and 27+- 2 uM. Of the monovalent and divalent cations studied, Zn2+ was the most effective divalent cation in catalyzing the formation of pyridoxal-5-phosphate from ATP and pyridoxal, whereas the enzymes had very little activity with Na+. The pH dependence of stability and activity were also examined. The stability of both enzymes was maximal in the 5.5 -7.5 pH range. The human enzyme had maximum catalytic activity in the narrow pH range of 5.5-6.0, while the activity of the porcine enzyme was maximal at pH 5.5. The physical interactions between PK and PLP-dependent enzymes (GAD and ALT) were demonstrated by fluorescence polarization. In addition, the temperature- and denaturant-induced denaturation of the two enzymes was studied by means of circular dichroism and fluorescence spectroscopy. Both enzymes showed an intermediate denaturation temperature (Tm): 48C for PPK and 46.4C for His-HPK. They also showed a markedly resistance against urea; the midpoint transition was 4.3 M for PPK and 3.45 M for His-HPK. On the contrary, both enzymes showed a weak resistance against GuHCl; the midpoint transition was 0.96 M for PPK and 1 M for His-HPK. In the second part of the project, five site-directed mutants were studied so as to examine the structure-function relationships of PK. The mutant enzymes including Y135A, Y135F, G240A, G242A, and H186A were expressed and purified to homogeneity. The H186A mutant had -70% residual activity, whereas the Y135F mutant exhibited less than 10% residual activity. The mutations in Y135A, G240A, and G242A completely abolished enzymatic activity. All mutants, except G240A, displayed similar CD spectra and fluorescence quenching result in comparison to the wild-type. The results of substrate binding analysis showed that mutation of tyrosine 135 to alanine (Y135A) or phenylalanine (Y135F), and mutation of glycine 242 and histidine 186 to alanine (G242A and H186A) affected pyridoxal binding, while the G240A mutant did not show any interaction with both pyridoxal and ATP. Taken together, these results suggested that Tyr135, Gly242, and His186 played a significant role in pyridoxal binding, and Gly240 seemed to be involved in maintaining the proper conformation of the protein rather than direct involvement in catalysis. In addition, the GuHCl-induced unfolding processes of the site-directed mutants were studied by circular dichroism and fluorescence spectroscopy. No dramatic change in the midpoint transition was observed, implying that the residues substituted were not essential for maintaining the protein conformational stability.

Files in this item

Files Size Format
b16974268.pdf 6.707Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.

     

Quick Search

Browse

More Information