Structural basis of acute intermittent porphyria and the relationship between mutations in human porphobilinogen deaminase and enzyme activity
Structural basis of acute intermittent porphyria and the relationship between mutations in human porphobilinogen deaminase and enzyme activity
This thesis deals with several human mutations that involve conserved arginine residues. Using molecular biology techniques, several recombinant human ubiquitous mutant porphobilinogen deaminases have been generated from cDNA specifying the enzyme and the recombinant mutant proteins have been overexpressed in E. coli.
Three arginine mutants, Arg 149 Gln, Arg 167 Gln and Arg 173 Gln were generated using PCR mutagenesis. Arg 149 Gln exhibits a CRIM-ve phenotype whereas Arg 167 Gln and Arg 173 Gln are examples of CRIM+ve mutations. The mutant deaminases have been investigated with respect to their specific activity, thermal stability and the presence of the dipyrromethane cofactor. Arg 167 Gln exhibits weak enzyme activity but is particularly interesting because it accumulates stable, partially assembled enzyme intermediate complexes. Both Arg 149 Gln and Arg 173 Gln mutants are essentially inactive, contain no dipyrromethane cofactor and exist as heat labile apo-enzymes.
Two other mutants, Arg 167 Trp and Trp 198 Ter, have also been investigated. The Arg 167 Trp mutant exhibits similar properties to the Arg 167 Gln mutant, showing weak enzyme activity and forming stable enzyme intermediate complexes. In contrast, the Trp 198 Ter mutant, the most common cause of AIP in Sweden, is an insoluble and completely inactive truncated protein.
Finally, studies have been initiated to crystallize and to determine the 3-dimensional structure of the Arg 167 Gln ubiquitous human deaminase mutant A preliminary X-ray structure at a resolution of 2.65 A, was obtained that shows only small differences from the E. coli enzyme, except for a large insertion in domain 3 that partially resolved. Some insight into substrate binding is obtained from the structure.
University of Southampton
Al-Dbass, Abeer M
bfed1ffc-b1d4-4305-83b0-52e7fcafed43
2001
Al-Dbass, Abeer M
bfed1ffc-b1d4-4305-83b0-52e7fcafed43
Al-Dbass, Abeer M
(2001)
Structural basis of acute intermittent porphyria and the relationship between mutations in human porphobilinogen deaminase and enzyme activity.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis deals with several human mutations that involve conserved arginine residues. Using molecular biology techniques, several recombinant human ubiquitous mutant porphobilinogen deaminases have been generated from cDNA specifying the enzyme and the recombinant mutant proteins have been overexpressed in E. coli.
Three arginine mutants, Arg 149 Gln, Arg 167 Gln and Arg 173 Gln were generated using PCR mutagenesis. Arg 149 Gln exhibits a CRIM-ve phenotype whereas Arg 167 Gln and Arg 173 Gln are examples of CRIM+ve mutations. The mutant deaminases have been investigated with respect to their specific activity, thermal stability and the presence of the dipyrromethane cofactor. Arg 167 Gln exhibits weak enzyme activity but is particularly interesting because it accumulates stable, partially assembled enzyme intermediate complexes. Both Arg 149 Gln and Arg 173 Gln mutants are essentially inactive, contain no dipyrromethane cofactor and exist as heat labile apo-enzymes.
Two other mutants, Arg 167 Trp and Trp 198 Ter, have also been investigated. The Arg 167 Trp mutant exhibits similar properties to the Arg 167 Gln mutant, showing weak enzyme activity and forming stable enzyme intermediate complexes. In contrast, the Trp 198 Ter mutant, the most common cause of AIP in Sweden, is an insoluble and completely inactive truncated protein.
Finally, studies have been initiated to crystallize and to determine the 3-dimensional structure of the Arg 167 Gln ubiquitous human deaminase mutant A preliminary X-ray structure at a resolution of 2.65 A, was obtained that shows only small differences from the E. coli enzyme, except for a large insertion in domain 3 that partially resolved. Some insight into substrate binding is obtained from the structure.
Text
814842.pdf
- Version of Record
More information
Published date: 2001
Identifiers
Local EPrints ID: 464501
URI: http://eprints.soton.ac.uk/id/eprint/464501
PURE UUID: cb4d636b-1927-43e8-a201-73e6c6b74e6a
Catalogue record
Date deposited: 04 Jul 2022 23:42
Last modified: 16 Mar 2024 19:33
Export record
Contributors
Author:
Abeer M Al-Dbass
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics