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, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.
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PMID:Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme. 21 11

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.
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PMID:Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis. 44 63

The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.
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PMID:Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group. 182 18

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.
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PMID:Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis. 218 28

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.
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PMID:ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin. 294 46

The half-life of mitochondrial adenosine triphosphatase and the relative rate constants of protein degradation for several fractions of rat liver have been measured by the double-isotope technique. It has been shown that the apparent turnover rates of some mitochondrial enzymes, far apart in size, such as carbamoyl phosphate synthetase, glutamate dehydrogenase and malate dehydrogenase, are not related to molecular weight or to size of subunits. In view of the possibility that mitochondrial proteins are degraded by different mechanisms, it was of interest to determine the half-life of a protein tightly bound to the inner membrane such as adenosine triphosphatase. The rate constants of degradation for rats fed a basal diet and injected at three-day intervals with isotopic leucine were: homogenate, kd = 0.195 days-1; mitochondria, kd = 0.135 days-1; cytosol, kd = 0.140 days-1; microsomes, kd = 0.28 days-1; ATPase, kd = 0.275 days-1. The rate constants of the cellular fractions of liver of rats fed a high protein diet did not change or showed a small increase, compared with those of animals fed the basal diet, while those from rats on the protein-free diet showed a decrease. The rate constant for adenosine triphosphatase showed an increase with high-protein and a decrease with protein-free diet. A procedure for the purification of ATPase from a single liver of a rat is described.
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PMID:Turnover of adenosine triphosphatase from rat liver mitochondria. Effect of high-protein and low-protein diets. 621 5

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.
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PMID:Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor. 622 15

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.
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PMID:Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate. 626 8

The activity and stability of carbamoyl-phosphate synthetase (EC 6.3.4.16) may involve hydrophobic and ionic bonds within the enzyme. The 1-anilino-8-naphthalene sulfonate (ANS) equilibrium binding method with hydrophobic and ionic sites in enzymes, therefore, seemed suitable for the study of the acetylglutamate activation and ATP binding of the enzyme. The enzyme had a high affinity for the dye but low fluorescent yields. The enzyme had 32-88 ANS binding sites, depending on combination with ATP and acetylglutamate, and individual affinity constants for each combination. Despite the large number of binding sites, the acetylglutamate and ATP concentrations for half-maximal fluorescent change (10-40 microM) corresponded to the high-affinity bound ATP (ATPB) and acetylglutamate Kd values. In kinetic studies, ANS competed with ATP or acetylglutamate. The extrapolated ANS Ki values for ATP or acetylglutamate were both 35 microM. This value agreed with the ANS Kd value of the enzyme X ATP conformation, indicating that this was the conformation competed for by ANS. Since ANS did not influence the HCO3-dependent ATPase, ANS was concluded to compete with the ATPB binding conformation and transitional changes. This study suggests that part of the activator role of acetylglutamate may be to change the tertiary structure of the enzyme to induce hydrophobic sites which are accessible to ANS and possibly at the ATPB site.
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PMID:Fluorescent probe study with 1-anilino-8-naphthalene sulfonate on acetylglutamate activation, ATP binding and conformational changes of the rat liver carbamoyl-phosphate synthetase. 687 Dec 32

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


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