Enzymes

1. ENZYMES

2. ENZYMES ENZYMES ALLOSTERIC ENZYMES ASPARTATE TRANSCARBAMOYLASE

3. ALLOSTERIC ENZYMES • ALLO – DIFFERENT • STERIC – SITE DEFINITION Allosteric are subset of enzymes that change their confirmational ensemble upon binding of an effector. i.e., allosteric enzymes binds other ligands at site other than the active site and can either be activated or inhibited.

4. EXAMPLES: • Fructose -1,6-bisphosphate (FBPase) • Phosphofructosekinase (PFKase) • Aspartatetranscarbamoylase (ATCase)

6. Introduction • Aspartatetranscarbamoylase (ATCase) is also called as aspartatecarbamoyltransferase. • E.C. no. - 2.1.3.2 • Organisms from the Archea, Prokarya and Eukarya kingdoms all produce ATCase. • ATCase catalyses the first step in a biosynthesis pathway that producesPYRIMIDINE NUCLEOTIDES. • ARTHUR PARDEE demonstrated the unique first step reaction of pyrimidine nucleotides pathway.

7. CLASSIFICATION • There are four major classes; • Class C – A homotrimer of catalytic chains each with a molecular mass of 34kDa. • Class 2 - The enyme from Bacillus subtiliswhich lack both homotrophic and heterotrophic properties. • Class A – These are 2nd form of ATCase which catalytes and regulates the enzyme. • Class B – These are best characterized class of ATCase comprised of two trimeric catalytic subunit of 34kDa polypeptides and three dimeric regulatory subunit of 17kDa polypeptides.

8. Structure of enzyme Redrawn and modified by ke.etal, in 1984. The class B is an allosteric enzyme, exhibiting both homotrophic and heterotrophic interaction. ATCase has 2 subunits C subunit (catalytic subunit;34kDa) and R subunit (regulatory;17kDa). The holoenzyme of ATCase is composed of 12 polypeptides chains,6 larger chains contain active site and 6 smaller chains contain inhibitor site with two separable catalytic trimer (C3) and three regulatory dimer(R2).

9. Pyrimidine biosynthesis • Pyrimidine biosynthesis occurs both in the body and through organic synthesis. • These is simpler process than the purines. • It is a de novo synthesis pathway . • End product of these pathway is Cytidinetriphosphate(CTP). Hence it undergoes feed back inhibition process. • Pyrimidine nucleotides are cytosine, thymine in DNA and uracil in RNA synthesis.

11. The allostericbehaviour of ATCase was investigated by John gerhartand Howard Schachmanin E-coli. • These reaction takes place in the cytosol of cell. • The condensation of carbamoyl phosphate along with aspartate to form N-carbamoylaspartate and release inorganic phosphate. • ATCase controls the rate of pyrimidine synthesis by altering its catalytic velocity in response to cellular levels of both pyrimidines and purines. • The feedback inhibition of ATCase controls pyrimidine biosynthesis.

12. The enzyme has two conformations, • R(relaxed state) active form, stable, binds substrate tightly. • T(tensed state) inactive form ,unstable ,In the absence of substrate . • ATCase switches between T and R state.

13. R and T conformations • In bacteria , the ATCase molecule consists of six catalytic subunits binds to substrate molecule and the allosteric subunits binds to allosteric inhibitor ,CTP. • The entire ATCase molecule, as well as its subunits exists in two conformations, active and inactive. • As CTP binds to regulatory subunits, they undergo a change in conformation, this change is transmitted to the catalytic subunits , which than also shift to an inactive form. • ATP prevents the changes induced by CTP because CTP is a allosteric inhibitor and ATP is a allosteric activator of these enzyme.

14. ATCase mechanism of enzyme action do not follow Michaelis and Menton curve It is a sigmoidal curve. ATCase is heterotrophically inhibited by CTP and heterotrophically activated by ATP. CTP therefore decreases the enzymes catalytic rate, whereas ATP increases it. Hence the ATP concentration required to activate ATCase is higher than the CTP concentration required to inhibit it by an equal amount.

15. Detection of enzyme • Analytical gel chromatography is used to detect the quaternary structure changes in both the holoenzyme and its catalytic subunit. • X-ray crystallography is used to determine the catalytic cycle and to obtained a snapshot. • Time-resolved small angle X-ray scattering is used to study the conformational changes of enzyme ATCase .

16. Physiological role of CTP and ATP Regulation of ATCase • CTP is a feed back inhibitor ; when CTP levels are high it is unnecessary to make more pyrimidines so the inhibition of ATCase slows down the pathway. when CTP levels fall, the inhibition is removed and more pyrimidines can be synthesized. • ATP is a purine nucleotide and is not a product of ATCase pathway. ATP is the major cellular energy source and if ATP levels are high, the cell is metabolically very active and preparing to divide . Therefore both ATP and CTP are needed for DNA synthesis. • Finally the role of the allosteric site is well illustrated by the example of enzyme called ASPARTATE TRANSCARBAMOYLASE .

17. REFERENCE • Principles of biochemistry; Lehninger • Biochemistry ; Donald Voet and Judieth G Voet • Biochemistry ; Palmer

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