ATCase.pptx

aspartate trascarbomylase

1. Enzyme model of Allosterism A typical example of enzyme model for allosterism is aspartate transcarbamoylase In E. coli, this enzyme is a multi-subunit protein complex composed of 12 subunits (300kDa in total) The composition of the subunits forms two trimers of catalytic subunits and 3 dimers of regulatory subunits The particular arrangement of catalytic and regulatory subunits in this enzyme affords the complex with strongly allosteric behaviour with respect to its substrates This enzyme is an archetypal example of allosteric modulation of the control of metabolic enzyme reactions

2. ATCase does not follow Michaelis-Menten kinetics but lies between low activity , low affinity “tight” or T and the high-activity high affinity “relaxed” or R states The binding of substrate to the catalytic subunits results in an equilibrium shift towards the R state 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

3. ATCase catalyses the committed step in pyrimidine biosynthesis ATCase controls the rate of pyrimidine biosynthesis by altering its catalytic velocity in response to cellular levels of both pyrimidines and purines . The end product of the pyrimidine pathway, CTP, induces a decrease in catalytic velocity

4. ATCase consists of two different kinds of polypeptide chains which have different roles The catalytic subunits catalyse the carbamoylation of the amino group of aspartate but do not have regulatory properties, while the regulatory subunits do not have any catalytic but contain the regulatory sites for effector binding. The ATCase holoenzyme is made of two catalytic trimers that are in contact and held together by three regulatory dimers

5. Each of catalytic domain is composed of two structural domains: the aspartate domain which contains most of the residue that bind to carbamoyl phosphate Each regulatory domain is also composed of two domains, the allosteric domain, which has the binding site for the nucleotide effectors the zinc domain consisting of four cysteine residues

6. Catalytic center Catalytic site of ATCase is located at the interface between two neighbouring catalytic chains in the same trimer and incorporates amino acid side-chains from both of these subunits. The rate enhancement of ATCase is achieved by orientation and stabilization of the substrates, intermediates and products rather than by direct involvement of amino acid residues in the catalytic mechanism

7. 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. ATP binds predominantly to the high-affinity sites and subsequently activates the enzyme UTP and CTP binding leads to inhibition of activity. UTP can bind to the allosteric site, but inhibition of ATCase by UTP is only possible in combination with CTP. With CTP present, UTP binding is enhanced and preferentially directed to the low affinity sites.

8. ENZYME REGULATION In biochemistry , allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site (that is, a site other than the protein's active site ).

9. Effectors that enhance the protein's activity are referred to as allosteric activators , whereas those that decrease the protein's activity are called allosteric inhibitors . The term allostery comes from the Greek allos ( ἄλλος ), "other", and stereos ( στερεὀς ), "solid (object)", in reference to the fact that the regulatory site of an allosteric protein is physically distinct from its active site. Allosteric regulations are a natural example of control loops, such as feedback from downstream products or feed forward from upstream substrates. Long-range allostery is especially important in cell signaling

10. Models of allosteric regulation Most allosteric effects can be explained by the concerted MWC model put forth by Monod , Wyman, and Changeux , or by the sequential model described by Koshland , Nemethy , and Filmer . Both postulate that enzyme subunits exist in one of two conformations , tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the tense state. The two models differ most in their assumptions about subunit interaction and the pre-existence of both states

11. Allosteric modulation Positive modulation Positive allosteric modulation (also known as allosteric activation ) occurs when the binding of one ligand enhances the attraction between substrate molecules and other binding sites. An example is the binding of oxygen molecules to hemoglobin , where oxygen is effectively both the substrate and the effector. The allosteric , or "other", site is the active site of an adjoining protein subunit . The binding of oxygen to one subunit induces a conformational change in that subunit that interacts with the remaining active sites to enhance their oxygen affinity

12. Negative modulation Negative allosteric modulation (also known as allosteric inhibition ) occurs when the binding of one ligand decreases the affinity for substrate at other active sites. For example, when 2,3-BPG binds to an allosteric site on haemoglobin , the affinity for oxygen of all subunits decreases. This is when a regulator is absent from the binding site

13. Types of allosteric regulation Homotropic A homotropic allosteric modulator is a substrate for its target enzyme , as well as a regulatory molecule of the enzyme's activity. It is typically an activator of the enzyme. For example, O 2 is a homotropic allosteric modulator of hemoglobin. Heterotropic A heterotropic allosteric modulator is a regulatory molecule that is not also the enzyme's substrate. It may be either an activator or an inhibitor of the enzyme. For example, H + , CO 2 , and 2,3-bisphosphoglycerate are heterotropic allosteric modulators of hemoglobin

14. Non-regulatory allostery A non-regulatory allosteric site refers to any non-regulatory component of an enzyme (or any protein), that is not itself an amino acid. For instance, many enzymes require sodium binding to ensure proper function. However, the sodium does not necessarily act as a regulatory subunit; the sodium is always present and there are no known biological processes to add/remove sodium to regulate enzyme activity. Non-regulatory allostery could comprise any other ions besides sodium (calcium, magnesium, zinc), as well as other chemicals and possibly vitamins.