Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.
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PMID:Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate. 1 11

The activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures. The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride. Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it. Such [14C]formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the [14C]formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme. When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of [14C]formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme. When reaction mixtures containing the enzyme, [gamma-32P]ATP, and [14C]bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate. Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate. Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.
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PMID:Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase. 18 54

Three catalytic domains of the Escherichia coli carbamoyl-phosphate synthetase (EC 6.3.5.5) have been identified in previous studies. These include the glutamine amide-N transfer domain in the carboxyl-terminal half of the glutaminase component and at least two adenine nucleotide binding sites in the synthetase component. To delineate the domains involved in subunit interactions, we have examined the effects of deletions and point mutations in the glutaminase and synthetase subunits on formation of the alpha beta holoenzyme. Deletion of the amino-terminal third of the glutaminase subunit abolishes interactions with the synthetase subunit, suggesting that this domain functions to stabilize the complex. Two subunit binding domains have been identified in the synthetase subunit. They are homologous to one another and are located in the amino-terminal and central regions of the synthetase component. These domains are adjacent to regions of the synthetase previously proposed to be involved in ATP binding and, possibly, activation of CO2. The new data enlarge the definition of the structural and functional domains in the two interdependent components of carbamoyl-phosphate synthetase.
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PMID:Escherichia coli carbamoyl-phosphate synthetase: domains of glutaminase and synthetase subunit interaction. 268 45

The urea biosynthetic pathway functions in mammalian liver to convert excess ammonia to urea and to maintain the concentration of ammonia in blood at nontoxic levels. This action is accomplished by enzymatic adaptation to quantitative changes in dietary protein. The first two enzymes of the pathway are found in the intestine of the adult mouse, but they do not adapt to dietary change. The enzymes in the intestine produce citrulline, which is carried by the bloodstream to the kidney, where it is converted by the next two enzymes of the pathway to arginine. This mechanism serves as the major source of circulating arginine. We have demonstrated that, at birth, the arginine-synthesizing enzymes in the kidney of the C57Bl/6 mouse are minimally developed, whereas in the intestine activity of carbamoyl-phosphate synthase is elevated and argininosuccinate synthase and lyase, usually present only in trace quantities in the adult intestine, are markedly increased in the newborn. The arginine formed cannot be converted to urea, since arginase does not appear in intestinal cells of the mouse until the age of 15 days. Except for liver, intestine has the most rapid protein turnover of any normal tissue. Our study indicates that, at a time when no other endogenous source of arginine for protein synthesis is available, the intestine of the newborn C57Bl mouse is capable of synthesizing arginine from either citrulline or NH3 and CO2.
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PMID:Development of arginine-synthesizing enzymes in mouse intestine. 372 68

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.
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PMID:Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase. 393 68

Synthesis of citrulline from ornithine, NH4+, and HCO3- by isolated pig liver mitochondria is inhibited by acetazolamide, a specific inhibitor of carbonic anhydrase, at the same concentrations which inhibit the mitochondrial matrix carbonic anhydrase. At an acetazolamide concentration sufficient to give complete inhibition of matrix carbonic anhydrase, the rate of citrulline synthesis is reduced by 71%, but no further decrease in citrulline is observed at higher concentrations of acetazolamide. Stimulation of O2 uptake by ornithine under conditions of maximal citrulline synthesis is also inhibited by acetazolamide. At pH 6.7, the ratio of citrulline synthesis is depressed relative to the rates observed over the range 7.2-7.7, and acetazolamide inhibits this rate by only 20%. These results support the hypothesis that the physiological role of carbonic anhydrase in liver mitochondria is to supply HCO3- as the substrate for the enzyme carbamoyl phosphate synthetase I, which provides the intermediate carbamoylphosphate in the rate-limiting step of citrulline synthesis. Since the uncatalyzed rate of CO2 hydration is rapid enough that it should not be rate-limiting for the carbamoylphosphate synthetase reaction, carbonic anhydrase appears to regulate access of HCO3- in the synthetase and so should be considered as one of the enzymes participating in the biosynthetic pathway leading to urea formation in the hepatocyte.
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PMID:Contribution of matrix carbonic anhydrase to citrulline synthesis in isolated guinea pig liver mitochondria. 640 83

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68

The NCE103 gene of the yeast Saccharomyces cerevisiae encodes a CA (carbonic anhydrase) that catalyses the interconversion of CO2 and bicarbonate. It has previously been reported that nce103 null mutants require elevated CO2 concentrations for growth in batch cultures. To discriminate between 'sparking' effects of CO2 and a CO2 requirement for steady-state fermentative growth, we switched glucose-limited anaerobic chemostat cultures of an nce103 null mutant from sparging with pure CO2 to sparging with nitrogen gas. This switch resulted in wash-out of the biomass, demonstrating that elevated CO2 concentrations are required even under conditions where CO2 is produced at high rates by fermentative sugar metabolism. Nutritional analysis of the nce103 null mutant demonstrated that growth on glucose under a non-CO2-enriched nitrogen atmosphere was possible when the culture medium was provided with L-aspartate, fatty acids, uracil and L-argininine. Thus the main physiological role of CA during growth of S. cerevisiae on glucose-ammonium salts media is the provision of inorganic carbon for the bicarbonate-dependent carboxylation reactions catalysed by pyruvate carboxylase, acetyl-CoA carboxylase and CPSase (carbamoyl-phosphate synthetase). To our knowledge, the present study represents the first full determination of the nutritional requirements of a CA-negative organism to date.
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PMID:Carbonic anhydrase (Nce103p): an essential biosynthetic enzyme for growth of Saccharomyces cerevisiae at atmospheric carbon dioxide pressure. 1594 16

A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.
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PMID:Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens. 2375 Sep 51

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO2; in the absence of CO2 dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO2. Dark CO2 fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO2 in the dark; in the lichen most dark CO2 fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of (14)CO2 in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO2 in the gas phase. Exogenous NH 4 (+) inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO2 but had no effect in the light. The overall data suggest that in the lichen dark CO2 fixation by the fungus may provide carbon skeletons which accept NH 4 (+) released by the cyanobacterium and that in the absence of CO2, NH 4 (+) directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.
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PMID:Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd. 2430 52


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