. Allostery and cooperativity. Aspartate transcarbamoylase (ATCase) is among the
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. Allostery and cooperativity. Aspartate transcarbamoylase (ATCase) is among the best nderstood regulatory enzymes. The reaction catalyzed by ATCase is briefly described n p.371 of Biochemistry Free for All. It is recommended that you look up the reaction n more detail for working the following problems. a) ATCase is an information-gathering enzyme because its speed needs to be adjusted according to the demand for its product molecule, N-carbamoyl-L-aspartate, shown below. What is this product of ATCase ultimately used for by the cell? b) How does ATCase sense that it should either speed up or slow down? c) The regulatory molecule that ATCase senses is structurally unlike ATCase's substrates and so the regulatory molecule is unlikely to bind specifically to ATCase's active site. Explain how the regulatory molecule interacts with ATCase. d) Describe the quaternary structural changes accompanying the regulation of ATCase. Use the nomenclature "T" and "R" to specify the structural states of the enzyme correlating with changes in the rate at which it catalyzes its reaction. (see p. 372).Explanation / Answer
a) Aspartate transcarbamoylase (ATCase) catalyzes the condensation of aspartate and carbamoyl phosphate to N-carbamoylaspartate and orthophosphate. This is the first step in synthesis of pyrimidines bases such as cytidine triphosphate (CTP) and thymidine triphosphate (UTP).
N-carbamoyl-aspartate is converted into dihydroorotic acid by Dihydroorotase enzyme. Dihydroorotic acid is converted to orotate by Dihydroorotate oxidase. Orotate phosphoribosyltransferase converts orotate and 5-Phospho-alpha-D-ribose 1-diphosphate to form Orotidine 5'-phosphate and Pyrophosphate. Orotidine-5-phosphate is decarboxylated by Orotidine-5'-phosphate decarboxylase to form UMP. UMP is converted to CTP or TMP.
Thus, the product N-carbamoylaspartate is used by the cell to synthesize pyrimidine bases CTP, UTP and TMP (or TTP). CTP and UMP are compounds that serve as cofactors in several metabolic reactions. Further, they are substrates in RNA synthesis.
b). ATcase is inhibited by CTP. When there is high concentration of CTP, the rate of the reaction is slow. At low CTP concentration, the rate of the ATcase reaction is high. This effect is known as end-product feedback inhibition. This occurs despite CTP being structurally different that than aspartate and carbomoyl phosphate. Hence, CTP will bind a site other than the active site, resulting in allosteric inhibition of ATcase. Adenosine triphosphate and guanosine triphosphate, which are formed by purine biosynthetic pathway, will act together to activate ATCase.
c) ATCase has a larger catalytic subunit that does not bind to CTP. However, the smaller regulatory (r) subunit, which has no catalytic activity, binds to CTP. ATCase has six catalytic chains surrounded by three pairs of regulatory chains. It is a dimer of a catalytic trimer and a trimer of regulatory dimer and can be represented as (c3)2(r2)3. Aspartate transcarbamoylase exists in two conformations: the inactive tense (T) state and the relaxed (R) state. CTP and UTP inhibit ATCase by weakening the R state. Similarly, ATP and GTP strengthen the R state and activate ATCase.
CTP binds to the interface between adjacent regulatory subunits. Each allosteric site has two subsites A and B bridged by Mg2+ ion. A subsite can bind CTP or ATP while B subsite can bind UTP or ATP. When CTP/UTP binds to the A regulatory sites, the relaxed R state is converted to an inactive tense T state. T state is converted to R state when UTP and ATP bind to the B subsite.
d) Positive co-operativity between catalytic sites for the binding of aspartate is due to association of C3 with r2. N-terminal regions of the regulatory chains R1 and R6 are present close to each other and the regulatory site. The C-terminal domain of the regulatory chains has a rubredoxin-like zinc-bound fold. When CTP and UTP bind, there is minor conformational change in catalytic subunit. However, in the regulatory subunit, the N-termini curl inward destabilizing the interface between adjacent subunits and disrupting the active R state.
In this inactive T state, the N-terminal backbones of Gln8 and Val9 interact with the UTP pyrimidine ring in B subsite of same regulatory subunit. ATP and GTP can similarly push the termini into the B subsites of the opposing regulatory subunits. As a result, the interface is locked supporting the R state.
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