Abstract
A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.
Highlights
Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP forming) EC 6.3.4.4) is essential to most organisms [1,2,3], catalyzing the first committed step in de novo biosynthesis of AMP, according to Reaction 1
A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site
The above provides a structural basis for ligand binding synergism, effects on kcat due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5-phosphoryl group of IMP
Summary
The kinetic mechanism is random sequential [4] with a preference for the binding of L-aspartate to the preformed enzymeIMP-GTP complex [5, 6]. In the second step of the reaction, the ␣-amino group of L-aspartate attacks the C-6 atom of 6-phosphoryl-IMP. The largest conformational change (ϳ9 Å) occurs in loop 40Ϫ53, which folds against the guanine nucleotide, putatively stabilizing the transition state by its interactions with Mg2ϩ and the phosphoryl groups of the nucleotide [12]. One complex (hereafter the pyrophosphate complex) has IMP, hadacidin (an analog of L-aspartate), and Mg2ϩ-pyrophosphate (in place of Mg2ϩ-GDP) at an ordered active site, demonstrating that the guanine base is not essential for organization of the active site. IMP-enzyme interactions alone suffice in driving the major conformational transitions of the synthetase active site. The above structures provide a basis for understanding ligand binding synergism in adenylosuccinate synthetase, substantial effects on catalytic rates due to mutations well removed from the active site, and the requirement for an unmodified 5Ј-phosphoryl group of IMP
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