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.