Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Abstract

Cytidine 5-triphosphate synthase catalyzes the ATPdependent formation of CTP from UTP using either NH 3 or L-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Glndependent CTP formation at concentrations of >0.15 mM and NH 3 -dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP ≈ 6-thio-GTP > ITP ≈ guanosine 5-tetraphosphate > O 6 -methyl-GTP > 2-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2,3-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5-triphosphate, 2-OH, and 3-OH are required for full activation. The 2-NH 2 group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH 2 group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH 3 -and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC 50 ≈ 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC 50 ≈ 4 -12 mM). 8-Oxoguanosine was the best inhibitor identified (IC 50 ‫؍‬ 80 M). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5-triphosphate synthase inhibitors.CTP synthase (CTPS 2 ; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)) catalyzes the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH 3 as the nitrogen source (Scheme 1) (1, 2). This glutamine amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer domain catalyzes the hydrolysis of Gln. The nascent NH 3 derived from this glutaminase activity is transferred via an NH 3 tunnel (3) to the N-terminal synthase domain, where it reacts with UTP that has been activated by ATP-dependent phosphorylation at the 4-position (4 -7). CTPS from Escherichia coli is the most thoroughly characterized CTPS with respect to its physical, kinetic, and structural properties. Indeed, E. coli CTPS serves as a good model for understanding catalysis by other CTPSs, including human CTPS (8), since CTPSs exhibit high conservation of functionally and structurally important residues and have few insertion/ deletion differences (3, 9).The de novo biosynthesis of pyrimidine nucleotides is highly regulated in E. coli, and consequently, CTPS is regulated in a complex fashion (1). GTP is required as a positive allosteric effector to increase the efficiency (k cat /K m ) of Gln-dependent CTP synthesis (10) by stabilizing the enzyme conformation that binds the tetrahedral intermediates formed during Gln ...

Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP

“…19,22,39,44 As previously observed, 19 the S 0.5 values of ATP and UTP are interdependent (Table 1). At subsaturating ATP concentration (150 μ M), the S 0.5 of UTP increased to 200 μ M, while at 60 μ M UTP, the ATP S 0.5 value increased to 330 μ M. Interestingly, we found that lowering the GTP concentration from the optimal (200 μ M) to its EC 50 (50 μ M) also reduced the ATP S 0.5 value to 81 ± 5 μ M ( n = 5).…”

“…Indeed, NADH inhibition resembles that of guanosine and other purines. 44,53 Like guanosine, NADH inhibits ammoniadependent synthesis (Figure S6) and similarly shifts both GTP activation and inhibition curves 10,44 (Figure 6e, compare to Figure 4 in ref 44). Potentiation of GTP dual effects is diagnostic for mutual and nonexclusive binding in noncooperative systems.…”

Characterization of filament‐forming CTP synthases from Arabidopsis thaliana

“…GTP was reported earlier as an activator of CTPS enzymes from E. coli (Lunn et al ., ; Barry et al ., ). Indeed, also the CTPS3 protein from Arabidopsis revealed markedly increased activity in the presence of GTP.…”

Regio‐Selective Chemical‐Enzymatic Synthesis of Pyrimidine Nucleotides Facilitates RNA Structure and Dynamics Studies

“…6.3.4.2). [6,22–27] All four enzymes were expressed and purified as described in our previous work. [28] …”