Aspartate carbamoyl transferase

Fig. 38. The zinc-thiolate cluster of the regulatory subunit of aspartate carbamoyl-transferase (Honzatko et al 1982). Coordinates are from the Brookhaven Protein Data Bank.

An even more striking instance of feedback control is found in the synthesis of UNA (see also Nucleoproteins and Nucleic Acids). As pointed nut in Ihat cnlry. normal UNA is composed of the nucleotides dcoxyguanosiiic. dcoxycytidine. deuxyadenosine. and thymidine, and Ihe amounts of the first and second of these are the same, as are those of the third and fourth. Obviously, close control is required of the amounts of these nucleotides that are synthesized by the cell, if they are to be made in the quantities required for DNA synthesis. Evidence has been found that ihe enzyme carbamoylphosphaie L-aspartate carbamoyl transferase, which... 

Allosteric Enzymes Typically Exhibit a Sigmoidal Dependence on Substrate Concentration The Symmetry Model Provides a Useful Framework for Relating Conformational Transitions to Allosteric Activation or Inhibition Phosphofructokinase Allosteric Control of Glycolysis Is Consistent with the Symmetry Model Aspartate Carbamoyl Transferase Allosteric Control of Pyrimidine Biosynthesis Glycogen Phosphorylase Combined Control by Allosteric Effectors and Phosphorylation... 

The reaction catalyzed by aspartate carbamoyl transferase, and the feedback inhibition of this enzyme in E. coli by the end product of the pathway, CTP. The series of small arrows represents additional... 

Aspartate Carbamoyl Transferase Allosteric Control of Pyrimidine Biosynthesis... 

Aspartate carbamoyl transferase, or aspartate trans-[j carbamylase, catalyzes the transfer of a carbamoyl I group... 

Effects of CTP, ATP, and mercurials on the rate of the reaction catalyzed by aspartate carbamoyl transferase. In the absence of CTP and ATP, the sigmoidal kinetics show positive cooperativity with respect to aspartate. CTP augments the positive cooperativity ATP reverses the effect of CTP. An organic mercurial, or ATP in the absence of CTP, eliminates the cooperativity, converting the curve from sigmoidal to hyperbolic. 

Subunit structure of aspartate carbamoyl transferase and the fragments produced by treating the enzyme with mercurials. In the complete enzyme (top), the three sets of regulator dimers are sandwiched between two trimers of catalytic subunits (see fig. 9.17). The approximate location of the active site in each c subunit of the trimer facing the viewer is indicated with a c. 

Y -phosphonacetyl-l.-aspartate (PALA) is structurally similar to a likely intermediate in the reaction catalyzed by aspartate carbamoyl transferase. The binding of PALA to the enzyme is prevented competitively by carbamoyl phosphate, and PALA prevents the binding of aspartate. These observations support the view that PALA binds at the catalytic site. 

Structures of aspartate carbamoyl transferase in the T conformation (a) and the R conformation (b) viewed along the threefold symmetry axis. The enzyme contains two cj clusters and three clusters. The a-carbon chains of one of the c3 groups are shown in aqua those of the other c3 group are in blue. One of the r subunits in each of the r2... 

The binding of PALA to the R conformation of aspartate carbamoyl transferase. Arg 105 and His 134 are provided by one domain of a c subunit, and Arg 167 and Arg 229 by the other domain. Ser 80 and Lys 84 are part of a loop of protein from a different c subunit. The PALA is indicated by red. The wavy green lines indicate polypeptide backbone structure. 

The main effect of AMP on either phosphorylase b or phosphorylase a is to decrease the Km for P,. This change can be interpreted as we have interpreted the actions of allosteric effectors on phosphofructokinase and aspartate carbamoyl transferase, on the model that the enzyme can exist in two conformational states (R and T) with different affinities for the substrate. However, phosphorylase presents the additional complexity that the equilibrium constant (L) between the two conformational states can be altered by a covalent modification of the enzyme. In the absence of substrates, [T]/[R] appears to be greater than 3,000 in phosphorylase b but to decrease to about 10 in phosphorylase a. 

Aspartate carbamoyl transferase (ACTase) from Escherichi coli has been studied in great detail. It catalyzes the first reaction unique to pyrimidine biosynthesis ... 

Si). The compound disrupts pyrimTdine syntheses by inhibition of aspartate carbamoyl transferase (ACT) (22). Concentrations of AAL-toxin of 10-50 ng/ml cause potent ACT inhibition. A microtechnique for the separation of these structurally related toxins has been achieved using HPLC (97). 

OH, NH2, SH groups glutamine synthetase plasmalemma ATPase coupling factor 1 mitochondrial membrane aspartate carbamoyl-transferase... 

Gouaux. J. E, and Lipscomb, W. N. 1440. Crystal structures ol phos-phonoacetamlde ligated I and phosphonoacetamide and inalonate ligated R states of aspartate carbamoyl transferase at 2.8-A resolution and neutral pH. BiochemistTy 24 .189-402. 

In mammals, carbamoyl phosphate synthetase II is the key regulatory enzyme in the biosynthesis of pyrimidine nucleotides. The enzyme is inhibited by UTP, the product of the pathway, and stimulated by purine nucleotides. In many bacteria, aspartate carbamoyl transferase is the key regulatory enzyme. It is inhibited by CTP and stimulated by ATP. 

EXAMPLE 5.5 Aspartate carbamoyl transferase, 310,000, catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate in the first committed step of pyrimidine biosynthesis (Chap. 14). The enzyme from the bacterium Escherichia coli consists of six subunits, three regulatory and three catalytic. CTP is a negative effector, i e., it inhibits the enzyme and does so through binding to the regulatory subunits. ATP is a positive effector that acts through the regulatory subunits, while succinate inhibits the reaction by direct competition with aspartate at the active site. 

The classic oligomeric protein, hemoglobin, is a dimer of dimers (Liddington et al., 1992). Other examples are octomeric mandelate recanase (Neidhart et al, 1991) and OePe dodecameric aspartate carbamoyl transferase (Stevens et al, 1990). 

Birds appear unable to synthesise any arginine via the urea cycle (see Section 5.4), which may be because of lack of carbamoyl phosphate synthetase I in mitochondria (Baker, 1991), and, as a result, the dietary requirement for arginine is higher than in growing mammals. However, they do appear to have a carbamoyl phosphate synthetase II in the cytosol (Maresh, Kwan Kalman, 1969). This may be part of the multienzyme protein G D (carbamoyl phosphate synthase-aspartate carbamoyl transferase-dihydroorotase), responsible for the biosynthesis of 3ihydroorotate, a pyrimidine precursor, but this is bound on the multienzyme protein, and it seems unlikely that it would be available for arginine biosynthesis (see Price Stevens, 1989). 

In addition to the above reactions of amino acids, two others involving specific amino acids must be mentioned. The first reaction involves the conversion of methionine to 5-adenosyl methionine by methionine adenosyl transferase. In this form, methionine is an important methyl donor in animal tissue. The second reaction is that involving aspartate carbamoyl transferase which converts L-aspar-tate to Ai -carbamoyl-I aspartate. This is the begiiming of a series of reactions culminating in the synthesis of pyrimidines. An intermediate step which forms orotidine-S-phosphate is catalysed by orotidine-5-phosphate pyrophosphoxylase. The orotidine-5-phosphate formed is the immediate precursor of uridine-S-phos-phate (UMP) which occupies a central position in pyrimidine synthesis. After some degree of transformation, UMP can be converted to cytosine-5-phosphate (CMP) or thymine-5-phosphate (TMP). It might be noted here that the purine... 

During pyrimidine synthesis, carbamoyl phosphate is utilized in a reversible reaction with the equilibrium shifted in favour of the synthesis of carbamoyl aspartate. The reaction is catalysed by aspartate carbamoyl-transferase (carbamoyl phosphate L-aspartate carbamoyltransferase, EC 2.1.3.2) which is widely distributed in nature. Rapidly growing tissues including various tumours are endowed with high levels of this enzyme... 

Regulation of P.b. In E. coli carbamoyl phosphate synthetase is activated by the purine nucleotides, IMP and XMP, and it is inhibited by the pyrimidine nucleotides, UMP and UDP. The key control point is the synthesis of N-carbamoylaspartic acid, catalysed by aspartate carbamoyl-transferase (aspartate trans-carbamylase, EC 2.1.3.2). In E. coli and Aerobacter aerogenes this enzyme is inhibited by CTP, and the inhibition is prevented by ATP. In Pseudomonas fluor-escens, the enzyme is inhibited by UTP, whereas in higher plants the regulatory inhibitor is UMP. 

Carbamoylphosphate L-aspartate carbamoyl-transferase Aspartate carbamyltransferase... 

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