Structure-function relationships of citrate synthase.

  • M. S. Robinson

Student thesis: Doctoral ThesisPhD

Abstract

The purification of citrate synthase from E. coli (wild-type) is described. The subunit Mr value was determined to be approximately 47,000 by SDS-polyacrylamide gel electrophoresis. The hexameric nature of this enzyme was established using bifunctional cross-linking reagents and analysis by SDS-polacrylamide gel electrophoresis. The purification of citrate synthase from a strain of E. coli containing elevated levels of the enzyme is also described. This organism possesses the qlt A gene on several copies of a hybrid plasmid and the structural and regulatory properties of its citrate synthase were found to be identical to the wild-type E. coli enzyme in all aspects studied. The implications of producing elevated levels of wild-type and mutant citrate synthase are discussed. Purification of citrate synthase from B. megaterium is reported. The enzyme was found to have a native Mr of 84,000 by a combination of analytical ultracentrifugation and gel filtration. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions revealed a subunit of Mr value 39,000 - 43,000. The enzyme was discovered to be dimeric by the use of cross-linking reagents and analysis by SDS-polyacrylamide gel electrophoresis, Modification of B. megaterium citrate synthase by chemical reagents was performed. The enzyme was insensitive to the thiol-specific reagent DTNB but inactivated by DEPC which specifically attacks histidine moieties. Spectroscopic analysis of the inactivated enzyme revealed 2 histidines modified per dimer necessary for a 100% loss in catalytic activity. Protection against inactivation by DEPC was afforded by both substrates and ATP. The purification of citrate synthase from a mutant strain of E. coli is described. The enzyme was found to have a native Mr value of 76,000 and a subunit Mr 42,000 - 43,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively. Comparisons of the structural and functional properties of citrate synthases from various sources are discussed in the light of the sum total of these findings.
Date of Award1984
Original languageEnglish
Awarding Institution
  • University of Bath

Cite this

Structure-function relationships of citrate synthase.
Robinson, M. S. (Author). 1984

Student thesis: Doctoral ThesisPhD