Kinetic Mechanism of Escherichia coli Asparagine Synthetase B

Abstract

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.

Rate-Limiting Steps and Role of Active Site Lys443 in the Mechanism of Carbapenam Synthetase

“…Adenylation and 4-membered ring formation occur followed by product release, PPi being the last to leave the active site (9,10). This kinetic picture agrees well with what is known about the progenitor asparagine synthetases, Class B (AS-B), a highly conserved family of primary metabolic enzymes that convert aspartate to asparagine (11). All three of these proteins proceed through an intermediate acyl-adenylate during the course of their individual reactions (Scheme 1).…”

Kinetic Characterization of Human Glutamine-fructose-6-phosphate Amidotransferase I

“…Solution of the crystal structures of bacterial PRPP-amidotransferase and asparagine synthetase revealed a hydrophobic channel that facilitates diffusion of ammonia from the glutaminase active site to the acceptor substrate site (43)(44)(45)(46)(47). Teplyakov et al (5,6) speculated that residues adjacent to the glutaminase active site of GFAT might represent the mouth of the ammonia channel.…”

Asparagine Synthetase Chemotherapy

“…The complex three-dimensional fold of the C-terminal domain of AS-B is similar to that observed in ATP pyrophosphatases (47), an enzyme superfamily that includes guanosine-5′-monophosphate synthetase (GMPS) (48), argininosuccinate synthetase (49), ATP sulfurylase (50), carbapenam synthetase (51), β-lactam synthetase (BLS) (52,53), and ThiI (54). Because all ATP pyrophosphatases convert ATP to AMP, the C-terminal active site of ASNS likely catalyzes activation of the side-chain carboxylate of aspartate to form an electrophilic intermediate, β-aspartyl-AMP (βAspAMP) 1, and inorganic pyrophosphate (PP i ) (Scheme 2) (28,55). As observed in other glutaminedependent amidotransferases (44,46,(56)(57)(58)(59)(60)(61), the two active sites of AS-B are linked by a solvent-inaccessible, intramolecular "tunnel" that is sufficiently wide to allow passage of an ammonia molecule (Figure 1b) (62).…”

“…As a result, recent work in this area has been concerned with obtaining a detailed understanding of (a) the steady-state kinetic mechanism for glutaminedependent asparagine production (55,63,64), and (b) critical intermolecular interactions that play a role in mediating the synthesis of βAspAMP and its subsequent reaction with ammonia (Scheme 2). Although the general properties of ASNS from a variety of sources appear very similar, many different kinetic mechanisms have been proposed for their glutamine-dependent synthetase activity (65)(66)(67).…”

“…Such a proposal is analogous to the kinetic mechanism of certain aminoacyl tRNA synthetases in which an acyl-AMP intermediate is synthesized prior to interaction of the enzyme with uncharged tRNA (69,70). The question of when glutamine or ammonia binds to AS-B was experimentally unresolved in these studies, however (55), and it was proposed that glutamine (and therefore ammonia) binds to the E.βAspAMP complex after PP i release. If correct, this order of catalytic events implies that βAspAMP must be stabilized within the synthetase site so as to prevent futile ATP hydrolysis prior to binding of the nitrogen source for asparagine production.…”