Nucleotide Binding to Trimeric dUTPase: Studies on Recombinant Enzymes using Kinetic and Spectroscopic Methods
(2000)- Abstract
- Recombinant dUTPase from the bacterium Escherichia coli, the retrovirus equine infectious anemia virus (EIAV) and Homo sapiens have been made available for physico-chemical characterisation. The protocols developed have also proven to be suitable for production of enzymes modified by isotope labelling or other methods. The improved accessibility of enzyme material has enabled the subsequent investigations presented in this thesis and elsewhere. Studies on EIAV dUTPase have assessed rate constants of ligand binding and catalysis, and provided new knowledge concerning the early phase of the catalytic reaction: the substrate binds with diffusion controlled rate (10<sup>8</sup> M<sup>-1</sup>s<sup>-1</sup>)... (More)
- Recombinant dUTPase from the bacterium Escherichia coli, the retrovirus equine infectious anemia virus (EIAV) and Homo sapiens have been made available for physico-chemical characterisation. The protocols developed have also proven to be suitable for production of enzymes modified by isotope labelling or other methods. The improved accessibility of enzyme material has enabled the subsequent investigations presented in this thesis and elsewhere. Studies on EIAV dUTPase have assessed rate constants of ligand binding and catalysis, and provided new knowledge concerning the early phase of the catalytic reaction: the substrate binds with diffusion controlled rate (10<sup>8</sup> M<sup>-1</sup>s<sup>-1</sup>) through a simple one-step mechanism. The C-terminal part of dUTPase, which contains similarities to phosphate-binding sequence motifs, is disordered in most crystal structures of the enzyme. This part of the viral enzyme is not involved in the substrate binding step. The dynamics of the C-terminus of the dUTPases from E. coli and EIAV were studied by NMR. The measurements showed a drastic reduction in flexibility upon binding of a substrate analogue to E. coli dUTPase. However, motion of the C-terminal "arm" of EIAV dUTPase could not be detected, possibly due to the rate of motion being below the NMR time range. Another method is needed if this difference between the two enzymes is to be clarified. Comparative studies of the kinetics of the three enzymes (E. coli, human and EIAV dUTPase) revealed that the viral enzyme is as efficient as the other dUTPases in hydrolysing the natural substrate dUTP. The similarities between the viral, bacterial and mammalian enzymes also include low values for <i>K<sub>M</sub></i> (0.1-1.1 µM) for dUTP and strict Michaelis-Menten kinetics. The EIAV dUTPase is less discriminatory than the cellular E. coli and human dUTPases against the related nucleotide dTTP. The binding of other nucleotides is also stronger relative to the affinity for the substrate. Some of the differences in binding strength could be attributed to structural differences between the enzymes. (Less)
- Abstract (Swedish)
- Popular Abstract in Swedish
I plan to add a summary in Swedish at the WWW-page www.biokem.lu.se/education/theses/johannordthesis.htm
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/40574
- author
- Nord, Johan LU
- supervisor
- opponent
-
- Docent Jonsson, Bengt-Harald, Department of Chemistry, Umeå university, Sweden
- organization
- publishing date
- 2000
- type
- Thesis
- publication status
- published
- subject
- keywords
- C-terminus, dUTPase, EIAV, Escherichia coli, flexibility, Homo sapiens, kinetics, NMR, nucleotide metabolism, rate constants of lig-and binding, production of recombinant enzyme, dUTP, specificity, Biochemistry, Metabolism, Biokemi
- pages
- 142 pages
- publisher
- Johan Nord, Department of Biochemistry, Center for Chemistry and Chemical Engineering, P.O. Box 124, S-221 00, Lund, Sweden
- defense location
- Hall B, Center for Chemistry and Chemical Engineering, Lund
- defense date
- 2000-05-26 13:15:00
- external identifiers
-
- other:ISRN: LUNKDL/(NKBK-1062)/1-142/2000
- ISBN
- 91-628-4133-5
- language
- English
- LU publication?
- yes
- id
- a0dc07e6-c343-48d7-b48c-3fd34ce9da4a (old id 40574)
- date added to LUP
- 2016-04-04 11:17:05
- date last changed
- 2018-11-21 21:03:50
@phdthesis{a0dc07e6-c343-48d7-b48c-3fd34ce9da4a,
abstract = {{Recombinant dUTPase from the bacterium Escherichia coli, the retrovirus equine infectious anemia virus (EIAV) and Homo sapiens have been made available for physico-chemical characterisation. The protocols developed have also proven to be suitable for production of enzymes modified by isotope labelling or other methods. The improved accessibility of enzyme material has enabled the subsequent investigations presented in this thesis and elsewhere. Studies on EIAV dUTPase have assessed rate constants of ligand binding and catalysis, and provided new knowledge concerning the early phase of the catalytic reaction: the substrate binds with diffusion controlled rate (10<sup>8</sup> M<sup>-1</sup>s<sup>-1</sup>) through a simple one-step mechanism. The C-terminal part of dUTPase, which contains similarities to phosphate-binding sequence motifs, is disordered in most crystal structures of the enzyme. This part of the viral enzyme is not involved in the substrate binding step. The dynamics of the C-terminus of the dUTPases from E. coli and EIAV were studied by NMR. The measurements showed a drastic reduction in flexibility upon binding of a substrate analogue to E. coli dUTPase. However, motion of the C-terminal "arm" of EIAV dUTPase could not be detected, possibly due to the rate of motion being below the NMR time range. Another method is needed if this difference between the two enzymes is to be clarified. Comparative studies of the kinetics of the three enzymes (E. coli, human and EIAV dUTPase) revealed that the viral enzyme is as efficient as the other dUTPases in hydrolysing the natural substrate dUTP. The similarities between the viral, bacterial and mammalian enzymes also include low values for <i>K<sub>M</sub></i> (0.1-1.1 µM) for dUTP and strict Michaelis-Menten kinetics. The EIAV dUTPase is less discriminatory than the cellular E. coli and human dUTPases against the related nucleotide dTTP. The binding of other nucleotides is also stronger relative to the affinity for the substrate. Some of the differences in binding strength could be attributed to structural differences between the enzymes.}},
author = {{Nord, Johan}},
isbn = {{91-628-4133-5}},
keywords = {{C-terminus; dUTPase; EIAV; Escherichia coli; flexibility; Homo sapiens; kinetics; NMR; nucleotide metabolism; rate constants of lig-and binding; production of recombinant enzyme; dUTP; specificity; Biochemistry; Metabolism; Biokemi}},
language = {{eng}},
publisher = {{Johan Nord, Department of Biochemistry, Center for Chemistry and Chemical Engineering, P.O. Box 124, S-221 00, Lund, Sweden}},
school = {{Lund University}},
title = {{Nucleotide Binding to Trimeric dUTPase: Studies on Recombinant Enzymes using Kinetic and Spectroscopic Methods}},
year = {{2000}},
}