A STUDY OF ESCHERICHIA-COLI ADENYLOSUCCINATE SYNTHETASE ASSOCIATION STATES AND THE INTERFACE RESIDUES OF THE HOMODIMER

The state of aggregation of adenylosuccinate synthetase from Escherich ia coli is a point of controversy, with crystal structures indicating a dimer and some solution studies indicating a monomer, Crystal struct ures implicate Arg(143) and Asp(231) in stabilizing the dimer, with Ar g(143) interacting directly with bound IMP of the a fold related subun it, Residue Arg(143) was changed to Lys and Leu, and residue Asp(231) was changed to Ala. Matrix-assisted laser desorption ionization mass s pectroscopy and analytical ultracentrifugation of the wild-type and th e mutant enzymes indicate a mixture of monomers and dimers, with a maj ority of the enzyme in the monomeric state, In the presence of active site ligands, the wild-type enzyme exists almost exclusively as a dime r, whereas the mutant enzymes show only slightly decreased dissociatio n constants for the dimerization, Initial rate kinetic studies of the wild-type and mutant enzymes show similar K-cat and K-m values for asp artate, However, increases in the K-m values of GTP and IMP are observ ed for the mutant, Changes in dissociation constants for IMP are compa rable with changes in K-m, values, Our results suggest that IMP bindin g induces enzyme dimerization and that two residues in the interface r egion, Arg(143) and Asp(231), play significant roles in IMP and GTP bi nding.