1887

Abstract

Citrate synthase catalyses the initial reaction of the citric acid cycle and can therefore be considered as the rate-controlling enzyme for the entry of substrates into the cycle. In , the specific activity of citrate synthase was found to be independent of the growth substrate and of the growth phase. The enzyme was not affected by NADH or 2-oxoglutarate and was only weakly inhibited by ATP (apparent = 10 mM). These results suggest that in neither the formation nor the activity of citrate synthase is subject to significant regulation. The citrate synthase gene, , was isolated, subcloned on plasmid pJC1 and introduced into . Relative to the wild-type the recombinant strains showed six- to eightfold higher specific citrate synthase activity. The nucleotide sequence of a 3007 bp DNA fragment containing the gene and its flanking regions was determined. The predicted gene product consists of 437 amino acids ( 48936) and shows up to 49.7% identity with citrate synthase polypeptides from other organisms. Inactivation of the chromosomal gene by gene-directed mutagenesis led to absence of detectable citrate synthase activity and to citrate (or glutamate) auxotrophy, indicating that only one citrate synthase is present in . Transcriptional analysis by Northern (RNA) hybridization and primer extension experiments revealed that the gene is monocistronic (1.45 kb mRNA) and that its transcription initiates at two consecutive G residues located 121 and 120 bp upstream of the translational start.

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1994-08-01
2021-10-23
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