Aspartate carbamoyltransferase (also known as aspartate transcarbamoylase or ATCase) catalyzes the first step in the pyrimidine biosynthetic pathway (). In ''

E. coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Es ...

'', the enzyme is a multi- subunit protein complex composed of 12 subunits (300 kDa in total). The composition of the subunits is C₆R₆, forming 2 trimers of catalytic subunits (34 kDa) and 3 dimers of regulatory subunits (17 kDa). The particular arrangement of catalytic and regulatory subunits in this enzyme affords the complex with strongly

allosteric In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site. The site to which the effector binds is termed the ''allosteric site ...

behaviour with respect to its substrates. The enzyme is an archetypal example of allosteric modulation of fine control of metabolic enzyme reactions. ATCase does not follow

Michaelis–Menten kinetics In biochemistry, Michaelis–Menten kinetics is one of the best-known models of enzyme kinetics. It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rat ...

. Instead, it lies between its low-activity, low-affinity "tense" and its high-activity, high-affinity "relaxed" states. The binding of substrate to the catalytic subunits results in an equilibrium shift towards the R state, whereas binding of CTP to the regulatory subunits results in an equilibrium shift towards the T state. Binding of ATP to the regulatory subunits results in an equilibrium shift towards the R state.

Reaction

ATCase is a highly regulated enzyme that catalyses the first committed step in pyrimidine biosynthesis, the condensation of l-aspartate and

carbamoyl phosphate Carbamoyl phosphate is an anion of biochemical significance. In land-dwelling animals, it is an intermediary metabolite in nitrogen disposal through the urea cycle and the synthesis of pyrimidines. Its enzymatic counterpart, carbamoyl phosphate s ...

to form N-carbamyl-L-aspartate and inorganic phosphate. The catalysis by ATCase serves as the rate limiting step in pyrimidine biosynthesis because it alters its catalytic velocity in response to cellular levels of both

pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The othe ...

s and

purine Purine is a heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which include substituted purin ...

s. The end-product of the pyrimidine pathway, CTP, decreases catalytic velocity, whereas ATP, the end-product of the parallel purine pathway, increases catalytic velocity.

Structure

The discussion of structure, catalytic center, and allosteric site that follows is based on the prokaryotic version of ATCase, specifically ''

s. Early studies demonstrated that ATCase consists of two different kinds of

polypeptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A ...

chains, which have different roles. The catalytic subunits catalyze the carbamylation of the

amino In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen atoms have been replaced by a substituent such ...

group of

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...

but do not have regulatory properties, while the regulatory subunits do not have any catalytic activity but contain the regulatory sites for effector binding. The ATCase

holoenzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. ...

is made of two catalytic trimers that are in contact and held together by three regulatory dimers, so the native form of the enzyme contains six chains of each type, with a total

molecular weight A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioche ...

of 310 kDa. Each of the catalytic domains is composed of two structural domains, the aspartate domain, which contains most of the residues responsible for binding

, and the carbamoyl phosphate domain, which contains most of the residues that bind to

. Each regulatory domain is also composed of two domains, the allosteric domain, which has the

binding site In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may includ ...

for the nucleotide effectors, and the

zinc Zinc is a chemical element with the symbol Zn and atomic number 30. Zinc is a slightly brittle metal at room temperature and has a shiny-greyish appearance when oxidation is removed. It is the first element in group 12 (IIB) of the periodic t ...

domain, consisting of four

cysteine Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, sometim ...

residues clustered in its C-terminal region. These residues

coordinate In geometry, a coordinate system is a system that uses one or more numbers, or coordinates, to uniquely determine the position of the points or other geometric elements on a manifold such as Euclidean space. The order of the coordinates is sig ...

atom Every atom is composed of a nucleus and one or more electrons bound to the nucleus. The nucleus is made of one or more protons and a number of neutrons. Only the most common variety of hydrogen has no neutrons. Every solid, liquid, gas, ...

that is not involved in any catalytic property, but has been shown to be essential for the association of regulatory and catalytic subunits. The three-dimensional arrangement of the catalytic and regulatory subunits involves several ionic and

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, t ...

stabilizing contacts between amino acid residues. Each catalytic chain is in contact with three other catalytic chains and two regulatory chains. Each regulatory monomer is in contact with one other regulatory chain and two catalytic chains. In the unliganded enzyme, the two catalytic trimers are also in contact.

Catalytic center

The catalytic site of ATCase is located at the interface between two neighboring catalytic chains in the same trimer and incorporates amino acid side-chains from both of these subunits. Insight into the mode of binding of substrates to the catalytic center of ATCase was first made possible by the binding of a bisubstrate analogue, N-(phosphonoacetyl)-L-aspartate (PALA). This compound is a strong inhibitor of ATCase and has a structure that is thought to be very close to that of the

transition state In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked wi ...

of the substrates. Additionally, crystal structures of ATCase bound to carbamoylphosphate and succinate have been obtained. These studies, in addition to investigations using site-directed mutagenesis of specific amino acids, have identified several residues that are crucial for catalysis, such as Ser52, Thr53, Arg54, Thr55, Arg105, His134, Gln137, Arg167, Arg229, Glu231, and Ser80 and Lys84 from an adjacent catalytic chain. The active site is a highly positively charged pocket. One of the most critical side-chains is from Arg54, which interacts with a terminal oxygen and the anhydride oxygen of carbamoyl phosphate, stabilizing the negative charge of the leaving phosphate group. Arg105, His134, and Thr55 help to increase the electrophilicity of the carbonyl carbon by interacting with the carbonyl oxygen. In general, the rate enhancement of ATCase is achieved by orientation and stabilization of substrates, intermediates, and products rather than by direct involvement of amino acid residues in the catalytic mechanism.

Allosteric site

The allosteric site in the allosteric domain of the R chains of the ATCase complex binds to the nucleotides ATP, CTP and/or UTP. There is one site with high affinity for ATP and CTP and one with 10- to 20-fold lower affinity for these nucleotides in each regulatory dimer. ATP binds predominantly to the high-affinity sites and subsequently activates the enzyme, while UTP and CTP binding leads to inhibition of activity. UTP can bind to the allosteric site, but inhibition of ATCase by UTP is possible only in combination with CTP. With CTP present, UTP binding is enhanced and preferentially directed to the low-affinity sites. On the converse, UTP binding leads to enhanced affinity for CTP at the high-affinity sites and together they inhibit enzyme activity by up to 95%, while CTP binding alone inhibits activity to 50% to 70%. Comparison of the crystal structures of the T and R forms of ATCase show that it swells in size during the allosteric transition, and that the catalytic subunits condense during this process. The two catalytic trimers move apart along the threefold axis by 12 Å, and they rotate about this axis by 5° each, ultimately leading to a reorientation of the regulatory subunits around their twofold axis by 15°. This

quaternary structure Protein quaternary structure is the fourth (and highest) classification level of protein structure. Protein quaternary structure refers to the structure of proteins which are themselves composed of two or more smaller protein chains (also refe ...

change is associated with alterations in inter-subunit and inter-domain interactions. The interaction between subunits C1-C4 and R1 is extensively modified during this conversion. In particular, there is large movement of amino acid residues 230-254, known collectively as the 240s loop. These residues are located at the cleft between the

and

domains at the C1-C4 interface. The overall outcome of these structural changes is that the two domains of each catalytic chain come closer together, ensuring a better contact with the substrates or their analogues. During this structural transition, some interactions between side-chains are lost and some others are established. Studies have confirmed that the position of the 240s loop directly affects substrate binding in the corresponding active site. Earlier studies using site-directed mutagenesis of the 240s loop showed that interactions between Asp271 and Tyr240, and between Glu239 of C1 and Tyr165 of C4 would stabilize the T-state, while interactions between Glu239 of C1 and both Lys164 and Tyr165 of C4 would stabilize the R-state. Located close to the 240s loop and the active site, the loop region encompassing residues 160–166 plays a role in both the internal architecture of the enzyme and its regulatory properties. In particular, the residue Asp162 interacts with Gln231 (known to be involved in aspartate binding), and binds the same residues in both the T and R states. A mutant that had this residue mutated to

alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side ...

showed a huge reduction in specific activity, a two-fold decrease in the affinity for

, a loss of homotropic cooperativity, and decreased activation by ATP. It was suggested that the change in the overall structure caused by the introduction of this residue affects other residues in the R1-C1, R1-C4 and C1-C4 interfaces, which are involved in the

transition.

Assembly of the complex

The regulatory and catalytic subunits exist as fused protein homologs, providing strong evidence that they would interact together. Two catalytic trimers and two regulatory dimers assemble to form an intermediate of aspartate carbamoyltransferase consisting of 6 catalytic subunits and 4 regulatory subunits.

References

External links

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