DOES ESCHERICHIA-COLI POSSESS A 2ND CITRATE SYNTHASE GENE

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-d eficient strain of E. coli (K114) has been mutated to generate a rever tant (K114r4) that produces a dimeric citrate synthase with altered ki netic and regulatory properties. On cloning and sequencing the gltA ge ne from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown t o be a catalytically essential residue in E. coli citrate synthase, an d we demonstrate that the hexameric enzyme produced on expression of t he gltA gene from K114 and K114r4 is inactive. The dimeric citrate syn thase from K114r4 has been purified and shown to be immunologically di stinct from the wild-type hexameric enzyme. Determination of its N-ter minal amino acid sequence demonstrates that the mutant citrate synthas e is encoded by a gene distinct from the E. coli gltA gene. The N-term inal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.