RESUMO
We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 +/- 0.2) to hyperbolic (Hill parameter = 1.0 +/- 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the ARg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319----Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R', by KCl.(ABSTRACT TRUNCATED AT 250 WORDS)