Studies on the regulatory and catalytic properties of E. coli citrate synthase

Handford, Penelope Ann (1986) Studies on the regulatory and catalytic properties of E. coli citrate synthase. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Two mutagenic strategies have been used to investigate E. coli citrate synthase function. An insertional mutation was introduced into the gltA gene by manipulation of a unique Clal restriction site. This was predicted to produce a change in reading frame such that a truncated protein was produced, containing the N-terminus of the native protein. The insertion mutation was successfully characterized at the genetic level using Southern blotting and DNA sequencing in conjunction with synthetic oligodeoxy-nucleotides. The combination of SDS-PAGE and use of polyclonal antibodies raised against the native protein failed, however, to identify the gene product. Reasons for the absence of gene product have been discussed. Site-directed mutagenesis of E. coli citrate synthase has been used to investigate the role of Asp362 in catalysis. This residue was converted to Gly362 and the remaining gene sequence checked for additional mutations using a series of synthetic oligodeoxynucleotides, spanning the structural gene. The mutated gene was subsequently recloned into pBR322 and the resulting plasmid, pCS31, used to transform a citrate synthase deletion mutant, W620, from which the mutant protein was successfully purified. The protein expressed < 2% of overall wild type enzyme activity and < 5% of wild type activity in each partial catalytic reaction measured. Binding of the substrates oxaloacetate and acetyl CoA to the mutant protein was investigated and revealed that acetyl CoA binding was severely reduced. Two mechanisms for the role of Asp362 in catalysis have been discussed. Catabolite repression of E. coli citrate synthase was studied using two different carbon sources for growth. The plasmid pDB2, containing the gltA gene, was used to transform W620, and the resulting strain DB1002 used for this study. The carbon source glycerol was found to repress citrate synthase production when compared with acetate, but in each case production was further repressed by the inclusion of glucose in the medium. (D74415/87)

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