Investigation of the GTP-binding consensus sequences in Escherichia coli adenylosuccinate synthetase and the enzyme's reaction mechanism
In this dissertation, on the basis of the similarities in sequence and structure between GTP-binding proteins and AMPSase, it was of interest to determine whether or not the GTP-binding consensus sequences have similar functions. It will be shown that the GTP-binding consensus sequences found in AMPSase play similar roles to those of the GTPase superfamily. The kinetic results show that Lys331 interacts with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry indicated that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the pre-existence of other substrates at the active site of the enzyme. The aspartate residue at position 333 of E. coli AMPSase is identified as a key determinant in the recognition of nucleoside triphosphates, and by modest changes in the side chain of the residue 333, the wild-type, GTP-hydrolyzing enzyme is transformed into an even more proficient XTP-hydrolyzing enzyme. A stoichiometric study of the metal ions in AMPSase is presented using kinetics to reveal, for the first time, that the second metal ion is involved in the reaction mechanism of AMPSase through complex formation with aspartate.