Studies on a Mutant Form of Escherichia coli Citrate Synthase Desensitised to Allosteric Effectors

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Abstract
Naturally-occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a large enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as E. coli, also by 2-oxoglutarate. Gram-positive bacteria and all eukaryotes produce a small citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies, the possibility of genetically altering one type of citrate synthase to the other was explored. By mutagenesis and suitable selection, a mutant of E. coli whose citrate synthase is both small and insensitive to NADH and 2-oxoglutarate was isolated. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.