CTP synthetase (CTPS) hydrolyzes glutamine and shuttles the resultant ammonia through an intermolecular tunnel to the point of incorporation with substrate UTP. Within the crystallized tetramer, <i>E. coli</i> CTPS subunits were seen to exist in two equally populated conformers that varied in the position of the side chain of H57. In one conformation, H57 is poised to hydrogen bond with substrate UTP and, in doing so, points away from the tunnel constriction; in the other conformation, H57 is pointed toward and potentially obstructs the ammonia passage. Thus, the conserved residue H57 is proposed to act as a “swinging gate”, coordinating the passage of nascent ammonia with the availability of acceptor-substrate UTP. To assess the potential role of this residue, we used site-directed mutagenesis to engineer H57A, H57N, H57L, and H57F and characterized the kinetics of each protein variant. Glutaminase activities remained largely unaffected by the mutations, whereas kcat/Km values for glutamine-dependent CTP synthesis reactions were decreased by nearly 100-fold. Thus, ammonia tunneling efficiencies were reduced to under 3%, compared to the ability of wild-type enzyme to incorporate 100% of nascent ammonia into the synthesis of CTP. Ammonia tunneling efficiences were unaffected by the V-type allosteric activator GTP. These results suggest that substitutions of the imidazole ring result in nascent ammonia being unproductively released into solvent, consistent with the role of H57 in directing ammonia to the synthetase domain. The synchronizing movement of H57 is hypothesized to result from its H-bonding interaction with substrate UTP; such a mechanism would suggest contributions by H57 to the binding affinity of UTP. However, UTP saturation profiles for all of the variants reveal Km values that are similar to or (in the case of H57A) less than that of wild-type CTPS.

Role of H57 in synchronizing ammonia transfer within E. coli CTP synthetase

249th National Meeting (2015)

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