Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Huang, Xinyi; Raushel, Frank M.

doi:10.1074/jbc.275.34.26233

Cited by 31 publications

(35 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…NH 2 OH and NH 2 NH 2 derived from the hydrolysis of Gln-OH and Gln-NH 2 , respectively) to test our hypothesis that the presence of an alanine at position 109 introduces a constriction in the NH 3 tunnel of E. coli CTPS. This approach has been used to demonstrate that the G359S mutant of CPS has a partially blocked NH 3 tunnel that prevents diffusion of NH 2 OH while still allowing some NH 3 to diffuse through [40]. The hypothesis that replacement of the bulky Leu109 by the smaller alanine could cause a tunnel blockage has precedent.…”

Section: Resultsmentioning

confidence: 99%

“…With the exception of Lieberman's work in 1956 [46], little is known about the ability of E. coli CTPS to utilize alternative NH 3 sources. In addition, some amidotransferases such as CPS [40] and asparagine synthase B [48] have been shown to hydrolyse Gln-OH and Gln-NH 2 to give rise to NH 2 OH and NH 2 NH 2 , respectively. Although E. coli CTPS has been shown to utilize Gln-OH as a substrate [16], the present study describes the first detailed kinetic characterization of the ability of E. coli CTPS to utilize alternative substrates.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Alternative substrates for wild‐type and L109A E. coli CTP synthases

Lunn

Bearne

2004

European Journal of Biochemistry

View full text Add to dashboard Cite

Cytidine 5¢-triphosphate (CTP) synthase catalyses the ATPdependent formation of CTP from uridine 5¢-triphosphate using either NH 3 or L-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH 3 that is generated is transferred via an NH 3 tunnel [Endrizzi, J.A., Kim CTP synthases to utilize NH 3 , NH 2 OH, and NH 2 NH 2 as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, c-glutamyl hydroxamate, and c-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of c-glutamyl hydroxamate and nascent NH 2 OH production from N 4 -hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH 3 tunnel of L109A, in the presence of bound allosteric effector guanosine 5¢-triphosphate, is not leaky but contains a constriction that discriminates between NH 3 and NH 2 OH on the basis of size.Keywords: amidotransferase; ammonia tunnel; CTP synthase; glutaminase; alternative substrates.Cytidine 5¢-triphosphate (CTP) synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH 3 as the nitrogen source [1,2]. This Gln amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer (GAT) domain utilizes a Cys-His-Glu triad to catalyse the rate-limiting hydrolysis of Gln (glutaminase activity) [3][4][5], and the nascent NH 3 derived from this glutaminase activity is transferred to the N-terminal synthase domain where the amination of a phosphorylated UTP intermediate is catalysed [6,7]. The reactions catalysed by CTPS are summarized in Scheme 1.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. As CTP has a central role in the biosynthesis of nucleic acids [8] and membrane phospholipids [9], CTPS is a recognized target for the development of antineoplastic agents [8,10], antiviral agents [10][11][12], and antiprotozoal agents [13][14][15]. The Escherichia coli enzyme is one of the most thoroughly characterized CTP synthases with respect to its physical and kinetic properties, and is regulated in a complex fashion [1]. GTP is required as a positive allosteric effector to increase the efficiency of the glutaminase activity and Gln-dependent CTP synthesis [3,16] but inhibits CTP synthesis at concentrations > 0.15 mM [17]. In addition, the enzyme is inhibited by the product CTP [18] and displays positive cooperativity for ATP and UTP [18][19][20]. ATP and UTP act synergistically to promote tetramerization of the enzyme to its active form [20].Recently, the X-ray crystal structure of E. coli CTPS was solved at a resolution of 2.3 Å [21]. The enzyme crystallised as a tetramer, presumably because of the high protein concentrations used as bound nucleotides were not present in the structure (i.e. apo-E. coli CTPS) [21]. The authors identified a solvent-fi...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Alternative substrates for wild‐type and L109A E. coli CTP synthases

Lunn

Bearne

2004

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The potentials were measured with a Ag/AgCl electrode and corrected to the standard hydrogen electrode. The concentrations of all species of the enzyme were calculated from the spectra and the molar absorptivities, and were plotted versus the equilibrium potential of the system using the Nernst equation (7), (7) where E cell is the measured cell potential, E°′ enzyme is the potential of the enzyme, [ox] and [red] are the concentrations of oxidized and reduced enzyme species, respectively.…”

Section: Reduction Potential Measurementsmentioning

confidence: 99%

“…glutamine, from one active site to a second where the ammonia is utilized in a second reaction. In the case of carbamoyl phosphate synthetases, carbamate is subsequently channeled to a third active site (7)(8)(9). 4-Hydroxy 2-ketovalerate aldolase/aldehyde dehydrogenase (acylating) channels a toxic acetaldehyde intermediate between two distant active sites located 25 Å from each other as a means of protecting the cell from the adverse effects of the intermediate before it is converted to acetyl coenzyme-A (10).…”

mentioning

confidence: 99%

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

et al. 2008

View full text Add to dashboard Cite

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, solved at sub-Ångstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To understand the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or the tunnel residues Phe359 or Gly347 to Trp or Asn, reduces the catalytic efficiency of oxidation. The K mO2 increases from 300 ± 35 μM for the wild-type enzyme to 617 ± 15 μM for the F359W mutant. The k cat for the F359W mutant catalyzed reaction decreases 13-fold relative to the wild-type catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Hydride transfer from the sterol to the flavin prosthetic group is no longer rate limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel-mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively are consistent with the cooperative kinetics. In the atomic resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is only observed in a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

show abstract

“…To test the validity of the molecular tunnel mapped out in the CPS structure, a series of site-directed mutant proteins were created within the ammonia tunnel and kinetically studied (24,25). One position in particular was targeted for analysis, namely Gly-359 of the small subunit, which forms an integral part of the interior wall of the putative molecular tunnel.…”

mentioning

confidence: 99%

Carbamoyl-phosphate Synthetase

Thoden

Huang

Raushel

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of ϳ100 Å in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.

show abstract

Restricted Passage of Reaction Intermediates through the Ammonia Tunnel of Carbamoyl Phosphate Synthetase

Cited by 31 publications

References 31 publications

Alternative substrates for wild‐type and L109A E. coli CTP synthases

Alternative substrates for wild‐type and L109A E. coli CTP synthases

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

Carbamoyl-phosphate Synthetase

Contact Info

Product

Resources

About