We examined the catalytic efficiency of 18 pig citrate synthase mutants. The residues mutated were selected according to two criteria: the conservation of that residue in all known titrate synthase sequences, and the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzyme and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetics of the enzyme. The mutations, as expected, affected the k(cat)s and K(m)s for OAA and acetyl-CoA to varying degrees. The catalytic efficiency of each of the mutants was determined by the k(cat)/K(m) for the individual substrates, OAA and acetyl-CoA. All mutations affected k(cat). There was only one mutant, Asp327Asn, in which the K(m)s primarily were affected. Most mutations affected both k(cat) and K(m) and included the following: His274Gly, His274Arg, Asp375Gly. Asp375Asn, Asp375Glu, Asp375Gln, His320Gly, His320Gln, His320Asn, His320Arg, Arg401His, Gly275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on k(cat) and K(m). The CS mutant Arg401Lys exhibited a modestly improved k(cat)/K(m) for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2.7 Å resolution of one of the mutants, His274Gly, have been undertaken. The mutant enzyme crystallizes in an 'open' conformation essentially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections observed between 6.0 and 2.7 Å resolution. The refined model revealed a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmutant, but otherwise the mutation does not result in major structural alterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of the citrate synthase catalyzed reaction may be an 'enolized' version of acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stabilize this by charge delocalization mediated by a hydrogen bond. These results provide the basis for further studies of the effects of these changes on the several reactive intermediates, activated substrates, and transition states which may occur along the reaction coordinate for this type of Claisen enzyme.
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