“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage |
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Authors: | Gregory D McCluskey Stephen L Bearne |
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Institution: | 1. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada;2. Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada |
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Abstract: | Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport. |
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Keywords: | CTP synthase Mutagenesis Molecular gate Ammonia tunnel Kinetics catalytic synchronization CD circular dichroism CPS carbamoyl phosphate synthase CTPS CTP synthase Gln H mean hydrodynamic diameter DON DLS dynamic light scattering GlmS glucosamine-6-phosphate synthase MD molecular dynamics NadE NTP nucleoside-5′-triphosphate 4 4 SD standard deviation |
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