ACTIVE-SITE MUTANTS OF PIG CITRATE SYNTHASE - EFFECTS OF MUTATIONS ONTHE ENZYME CATALYTIC AND STRUCTURAL-PROPERTIES

We examined the catalytic efficiency of 18 pig citrate synthase mutant s. The residues mutated were selected according to two criteria: the c onservation of that residue in all known citrate synthase sequences, a nd the importance of that residue in substrate-amino acid interactions suggested by the extensive crystal structure information on the enzym e and its complexes. Several changes were made at certain residues to probe the effects of size, hydrogen bonding, and charge on the kinetic s 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 efficie ncy of each of the mutants was determined by the k(cat)/K-m for the in dividual substrates, OAA and acetyl-CoA. All mutations affected k(cat) . There was only one mutant, Asp327Asn, in which the K(m)s primarily w ere 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, Gly 275Val, and Gly275Ala. The mutations, Arg401Gly, Arg401Lys, His235Gln, and Asn242Glu, had smaller effects on k(cat) and K-m. The CS mutant A rg401Lys exhibited a modestly improved k(cat)/K-m for both substrates compared to the nonmutant enzyme. X-ray crystallographic studies at 2. 7 Angstrom resolution of one of the mutants, His274Gly, have been unde rtaken. The mutant enzyme crystallizes in an ''open'' conformation ess entially isomorphous to wild type. The refined model has good geometry and a crystallographic R factor of 0.187 for 11 441 reflections obser ved between 6.0 and 2.7 Angstrom resolution. The refined model reveale d a localized relaxation of the structure to relieve strain imposed by a high-energy main and side chain conformation of His274 in the nonmu tant, but otherwise the mutation does not result in major structural a lterations. Preliminary electrostatic calculations provide support for the concept that the transition state in the rate-limiting step of th e citrate synthase catalyzed reaction may be an ''enolized'' version o f acetyl-CoA that is neither neutral nor fully negatively charged and that a possible role for the catalytically essential His274 is to stab ilize 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 fo r this type of Claisen enzyme.