Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase.

111

171

Two proteins essential for the biosynthesis of the amino acid histidine in Escherichia coli have been overexpressed and purified to apparent homogeneity. The protein encoded by the hisF gene has an ammonia-dependent activity that results in the conversion of the biosynthetic intermediate N1-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4- carboxamide ribonucleotide (PRFAR) to imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamido-1-beta-D- ribofuranosyl 5'-monophosphate (AICAR). The second protein encoded by the hisH gene exhibits no detectable catalytic properties with biosynthetic intermediate PRFAR, glutamine, or ammonia. In combination, the proteins are capable of a stoichiometric conversion of glutamine and PRFAR to form AICAR, IGP, and glutamate. Neither protein alone is capable of mediating a conversion of the nucleotide substrate to a free metabolic intermediate. The HisH and HisF proteins form a stable 1:1 dimeric complex that constitutes the IGP synthase holoenzyme. Steady-state kinetic parameters for the holoenzyme indicate that glutamine is a more efficient substrate relative to ammonium ion by a factor of 10(3). The HisF subunit will support an ammonia-dependent reaction with a turnover number similar to that of the holoenzyme with glutamine. The glutaminase activity for the holoenzyme is 0.8% of that in the presence of the nucleotide substrate PRFAR. There are critical subunit interactions that mediate the catalytic properties for glutamine hydrolysis. The catalytic turnover of glutamine can be increased up to 37-fold by the addition of either the product IGP or the biosynthetic precursor N1-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (5'-ProFAR). The mechanistic significance of this glutaminase activity compared to other trpG type glutamine amidotransferases is discussed.

Section: Discussionmentioning

confidence: 99%

Imidazole glycerol phosphate synthase: The glutamine amidotransferase in histidine biosynthesis

Vj²

1993

111

171

“…However, in most purifications of the truncated enzymes, the protein from the protamine sulfate precipitation step was filtered and directly applied to the Waters Protein-Pack column. The protein concentration was determined using an absorbance coefficient of Aaso -0.69/mg (Rubino et al, 1986).…”

Section: R1031amentioning

confidence: 99%

Regulatory Changes in the Control of Carbamoyl Phosphate Synthetase Induced by Truncation and Mutagenesis of the Allosteric Binding Domain

Czerwiński¹,

Mareya²,

Raushel³

1995

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and two molecules of MgATP. The enzyme is composed of two nonidentical subunits. The small subunit catalyzes the hydrolysis of glutamine to glutamate and ammonia. The large subunit catalyzes the formation of carbamoyl phosphate and has the binding sites for bicarbonate, ammonia, MgATP, and the allosteric ligands IMP, UMP, and ornithine. The allosteric ligands are believed to bind to the extreme C-terminal portion of the large subunit. Truncation mutants were constructed to investigate the allosteric binding domain. Stop codons were introduced at various locations along the carB gene in order to delete amino acids from the carboxy-terminal end of the large subunit. Removal of 14-119 amino acids from the carboxy-terminal end of the large subunit resulted in significant decreases in all of the enzymatic activities catalyzed by the enzyme. A 40-fold decrease in the glutamine-dependent ATPase activity was observed for the delta 14 truncation. Similar losses in activity were also observed for the delta 50, delta 65, delta 91, and delta 119 mutant proteins. However, formation of carbamoyl phosphate was detected even after the deletion of 119 amino acids from the carboxy-terminal end of the large subunit. No allosteric effects were observed for UMP with either the delta 91 or delta 119 truncation mutants, but alterations in the catalytic activity were observed in the presence of ornithine even after the removal of the last 119 amino acids from the large subunit of CPS. Six conserved amino acids within the allosteric domain were mutated.(ABSTRACT TRUNCATED AT 250 WORDS)

“…Incubation at 25 °C for specified times was carried out upon addition of 10-40 Mg of CPS. Reactions were quenched by adding an acid reagent and diacetyl monoxime (Rubino et al, 1986). The citrulline concentration was measured from the absorbance at 464 nm using an absorption coefficient of37 800…”

Section: C248smentioning

confidence: 99%

A Molecular Wedge for Triggering the Amidotransferase Activity of Carbamoyl Phosphate Synthetase

Mareya¹,

Raushel²

1994

The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis. Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions. Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit. In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate. Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues. These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide. Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme. The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W. There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS)