CTP Synthetase (CTPS) catalyzes glutamine hydrolysis (GATase) in one domain, releasing ammonia to serve as a nucleophile in a distant amidoligase (ALase) domain. Crystal structures have identified a tubular passage between the GATase and ALase sites, highlighting a possible route for nascent ammonia. P54 and V60 create the narrowest constriction of this passage. GTP serves as an allosteric activator of the enzyme, enhancing GATase activity and, perhaps, optimizing the conformation of the ammonia passage. We seek to assess the validity and GTP-dependence of the proposed molecular passage. Our method is to replace P54 and V60 with smaller (P54A, V60A) and larger (P54L, V60L) residues and monitor the dependence of their GATase and CTP synthesis activities on GTP concentration. The ratio of CTP synthesis to GATase activity defines the coupling parameter (CP) between active sites. Mutation of P54 dramatically reduces both CTP synthesis and GATase activities. These data highlight P54 as integral to structure/function but unsuitable as a target for the systematic, engineered blockage of the proposed ammonia passage. By contrast, V60L exhibits normal GATase activity and a CP value of 0.84 when GTP is saturating. In the absence of GTP, the value of CP is reduced to 0.09. By comparison, wild-type enzyme and V60A maintains perfect coupling (CP=1) with and without GTP. These data are consistent with the V60L mutation projecting a larger R-side chain into the passage and partially obstructing ammonia delivery, and GTP allosterically organizing the ammonia passage to widen it beyond the V60L obstruction.

V60L uncouples glutaminase and synthetase activities in CTP synthetase:  GTP partially restores coupling

243rd National Meeting (2012)

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