Chapter 10 Regulatory strategies

1. Chapter 10 Regulatory strategies

2. Like motor traffic, metabolic pathways flow more efficiently when regulated by signals.

3. Enzymatic activity is regulated in five principal ways : • Allosteric control. Allosteric proteins contain distinct regulatory sites and multiple functional sites. cooperativity • Multiple forms of enzymes. Isozymes, or isoenzymes, provide an avenue for varying regulation of the same reaction at distinct locations or times. • Reversible covalent modification. The catalytic properties of enzymes are altered by the covalent attachment of a modifying group. Kinase - phosphatase • Proteolytic activation : Irreversible convert an inactive enzyme into an active one. • Controlling the amount of enzyme present. Transcription level

4. 10.1 Aspartate transcarbamoylase is allosterically inhibited by the end product of its pathway Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines(such as cytidine triphosphate CTP).

5. ATCase is inhibited by CTP, the final product of the ATCase-initiated pathway. • The inhibition of ATCase by CTP is an example of feedback inhibition, the inhibition of an enzyme by the end product of the pathway. • CTP is structurally quite different from the substrate and product of the reaction. • Thus CTP must bind to a site distinct from the active site (allosteric or regulatory site).

6. Allosterically regulated enzymes do not follow Michaelis-Menten kinetics • The curve differs from that expected for an enzyme that follows Michaelis-Menten kinetics. • Sigmoidal kinetics • In hemoglobin, sigmoidal curve from cooperativity

7. ATCase consists of separable catalytic and regulatory subunit • ATCase can be separated into regulatory(r) and catalytic(c) subunits ; how to know? evidence? • p-hydroxymercuribenzoate reacts with sulfhydryl group of cystein residues in ATCase resulting in separation of subunits.

8. Mercurials dissociate ATCase into two kinds of subunit. How? • The subunits separated by ion-exchange chromatography or by ultracentrifugation in a sucrose density gradient. • The larger subunit : catalytic subunit(c). No CTP binding and no sigmoidal kinetics • The smaller subunit : regulatory subunit(r). No catalytic activity • 2 c3(trimer) + 3 r2(dimer) → c6r6

9. Allosteric interactions in ATCase are mediated by large changes in quaternary structure 1RAI.pdb • Two catalytic trimers are stacked one on top of the other, • linked by three dimers of the regulatory chains. • How to mercurial compound dissociate ATCase into two kinds of subunit? – zinc bound to four cysteines, destabilize r-subunits

10. 2IPO.pdb PALA : bisubstrate (two substrates) analog, competitive inhibitor of ATCase. binds to the active sites and blocks ATCase. ※ ACTase was crystallized in the presence of PALA.

11. PALA PALA binds at sites lying at the bounderies between pairs of c chains within a catalytic trimer.

12. - Conformational change : catalytic trimers move 12Å farther apart, rotate 10 degrees about their threefold axis of symmetry and dimers rotate 15 degrees when PALA(substrate analog) bound. In the absence of substrate T(tense) state In the presence of substrate R(relaxed) state

13. The enzyme exists in an equilibrium between the T and R state. • In the absence of substrate, almost all the enzyme molecules are in the T state(low affinity for substrates and low catalytic activity). • Occasional substrate binding to active site → entire enzyme shifts to the R state(higher binding affinity).

14. Mechanism for allosteric regulation 1). Concerted mechanism - the change in the enzyme is ‘all or none’ (explains the behavior of ATCase well) 2). Sequential model - the binding of ligand to one site on the complex can affect neighboring sites without T-toR- transition ※ Most other allosteric enzymes have features of both models.

15. High Km low Km • - Mixture of two Michaelis-Menten enzymes. • As the concentration of substrate is increased, the equilibrium shifts from the T state to the R state. • Homotrophic effect by substrate; benefical when small change in substance conc is physiologically important

16. Allosteric regulators modulate the T-to-R equilibrium • - The enzyme is in the T state when bound to CTP. • Binding sites for CTP exist in regulatory chains which are far away from each active sites. • How can CTP inhibit the catalytic activity of the enzyme?

17. In the absence of any substrate -CTP stabilize the T state. -The binding of CTP shifts the equilibrium toward the T state and makes it more difficult for substrate binding to convert the enzyme into the R state.

18. In presence of CTP, more substrate is required to attain a given reaction rate. In presence of ATP, the reaction rate is increased. High levels of ATP prevent CTP from inhibiting the enzyme. Nonsubstrate effects on allosteric enzyme Heterotropic effects

19. Physiological roles of the increase in ATCase activity in response to increased ATP concentration 1) High ATP concentration signals a high concentration of purine nucleotides in the cell. (to balance between purine and pyrimidine) 2) A high concentration of ATP indicates that energy is available for mRNA synthesis and DNA replication and lead to the synthesis of pyrimidines needed for these processes.

20. Equilibrium between the T and the R state R ↔ T L = [T]/[R] L = the equilibrium constant between the R and the T forms CTP-saturated form, the value of L increases from 250 to 1250. For the ATP saturated form, the value of L decreases to 70.

21. 10.2 Isozymes provide a means of regulation specific to distinct tissues and developmental states • Isozymes (or isoenzymes) are enzymes that differ in amino acid sequence yet catalyze the same reaction. - have Different Km, different regulatory molecules

22. LDH(lactate dehydrogenase): catalyzes a step in anaerobic glucose metabolism and glucose synthesis (pyruvate  lactate ch16 참조). • H isozyme : expressed in heart muscle. • M isozyme : expressed in skeletal muscle. • amino acid sequences are 75% identical each other. • H isozyme (square) has higher affinity for substrate than M (circle) The rat heart LDH isozymeprofile changes in the course of development. Functional enzyme is tetrameric.

23. 1. Sequential displacement - Ordered. Lactate dehydrogenase ① ② ① ② - The coenzyme always binds first and the lactase is always released first.

24. LDH isozyme content varies by tissue. -The M4 isozyme functions optimally in the anaerobic environment of skeletal muscle, whereas the H4 isozyme does in the aerobic environment of heart muscle. Myocardial infarction, heart attack; H4 to H3M ratio increase in blood

25. 10.3 Covalent modification is a means of regulating enzyme activity Some modifications are reversible.

26. Phosphorylation is a highly effective means of regulating the activities of target proteins • - 30% of eukaryotic proteins are phosphorylated. • The enzymes catalyzing phosphorylation reactions are called protein kinases. • ATP is the most common donor of phosphoryl group. γ β α

28. Protein phosphatases reverse the effects of kinases by catalyzing the removal of phosphoryl groups attacked to proteins. • Hydroxyl-containing side chain is regenerated and Pi is produced. • Vital role in cells because the enzymes turn off the signals • One class of highly conserved phosphatase called PP2A suppresses the cancer-promoting activity of certain kinases.

29. Kinase Phosphatase • - The phosphorylation and dephosphorylation are not the reverse of one another; irreversible under physiological conditions without enzymes • With only the help of kinases and phosphatase, take place • The rate of cycling between the phosphorylated and the dephosphorylated states depends on the relative activities of kinases and phosphatases

30. Phosphorylation is a highly effective means of controlling the activity of proteins for several reasons; 1) A phosphoryl group adds two negative charges to a modified protein. New electrostatic interactions 2) A phosphoryl group can form three or more hydrogen bonds. 3) The free energy of phosphorylation is large. (-12kcalmol-1 by ATP, half is consumed in making phosphorylation; half (6kcalmol-1) is conserved in the phosphorylated protein. Phosphorylation can change the conformational equilibrium by the order of 104 ; 1.4kcalmol-1 correspond to 10 fold increase in an equilibrium constant) 4) Phosphorylation and dephosphorylation can take place in less than a second or over a span of hours. 5) Phosphorylation often highly amplify signaling effects. 6) ATP is the cellular energy currency. Links the energy status of the cell to the regulation of metabolism.

31. Cyclic AMP activates protein kinase A (PKA) by altering the quaternary structure • Flight or fight; epinephrine (adrenaline) triggers cAMP formation • Cyclic AMP subsequently activates a key enzyme: protein kinase A. • The kinase alters the activities of target proteins by phosphorylating specific serine or threonine residues. The consensus sequence recognized by PKA is R-R-X-S/T-Z ; (X, small residues; Z, large hydrophobic one)

32. -PKA provides a clear example of the integration of allosteric regulation and phosphorylation. • PKA consists of two kinds of subunit: a 49kDa regulatory(R) and a 38kDa catalytic(C) subunit. In the absence of cAMP, R and C form complex. Pseudosubstrate sequence inhibit catalytic subunit. In the presence of cAMP, R and C dissociate from complex. Catalytic subunit is activated.

33. ATP and the target protein bind to a deep cleft in the catalytic subunit of protein kinase A • The 350-residue catalytic subunit has two lobes. • ATP and part of the inhibitor fill a deep cleft between lobes. • The two lobes move closer to one another on substrate binding.

34. The pseudosubstrate seq. : Arg-Arg-Asn-Ala-Ile - Arg(pseudosubstrate) and Glu(PKA) form an ion pair.- Ile(pseudosubstrate) hydrophobically interact with leu(KPA)

35. 10.4 Many enzymes are activated by specific proteolytic cleavage Specific proteolysis is a common means of activating enzymes and other proteins in biological systems.Ex) ; 1) Digestive enzymes 2) Blood clotting 3) Hormones; proinsuline 4) Collagen; procollagen 5) Developmental processes: procollagenase (tadpole, 출산) 6). Programmed cell death, apoptosis: procaspases The inactive precursor is called a zymogen or a proenzyme.Proteolytic activation occurs just once.

36. Chymotypsinogen is activated by specific cleavage of a single peptide bond • Chymotrypsin : digestive enzyme in the small intestine. • Synthsized as inactive precursor, chymotrypsinogen. • Enzymes are synthesized in the acinar cells of the pancreas and stored inside membrane-bounded granules. • The cell is stimulated by hormone or nerve, the granules are released into a duct leading to the duodenum (십이지장)

37. Chymotrypsinogen • Single polypeptide of 245 amino acids is cleaved by trypsin. Linked by interchain disulfide bonds

38. Proteolytic activation of chymotrypsinogen leads to the formation of a substrate-binding site • The cleavage of the peptide bond between amino acid 15 and 16 triggers key conformational changes. • Alteration of the position of Ile16. • The electrostatic interaction between Ile16 and Asp 194 is essential for the structure of active chymotrypsin. • Substrate binding cavity and the oxyanion hole are formed after proteolytic cleavage.

39. The generation of typsin from trypsinogen leads to the activation of other zymogens from the duodenum cells (십이지장) Cleave lysine-isoleucine peptide - The formation of trypsin by enteropeptidase is the master activation step.

40. Some proteolytic enzymes have specific inhibitors Pancreatic trypsin inhibitor : - 6kda - Inhibits trypsin by binding- very tightly to its active site. Turnover rate is very low - Lys 15 interact with specificity pocket of trypsin (Asp189). Trypsin inhibition → prevention of tissue damage of pancreas or pancreatic ducts.

41. ※ α1-Antitrypsin: protease inhibitor of elastase, a secretary product of neurophils. - Block the elastases by binding to the active sites nearly irreversibly. Antielastase? - Genetic disorder of α1-Antitrypsin : emphysema (low α1-Antitrypsin concentraion in the serum – only 15% → cannot inhibit elastase → elastase destroys alveilar walls in the lung) Oxidation of methionine 358 of inhibitor (by smoking) → cannot bind to elastase → lung damage

42. Blood clotting is accomplished by a cascade of zymogen activations • Blood clots are formed by a cascade of zymogen activations. Amplification! • rapid response to trauma • After the tissue factor is exposed, small amounts of thrombin, the key protease in clotting, are generated. • Thrombin activates enzymes and factors that lead to the generation of yet more thrombin (= positive feedback) • 혈우병? Exposure of anionic surfaces Integral membrane glycoprotein Signal amplified Transglutaminase

43. Fibrinogen is converted by thrombin into a fibrin clot Rod : triple-stranded α–helical coiled coil 6 chain = 340kda • - Fibrinogen: is made up of three globular units connected by two rods. • Thrombin cleaves four arg-gly peptide bond in the central region of fibrinogen. • The cleaved peptide of Aα and Bβ chain = fibrinopeptide • -result in (αβγ)2

44. Fibirin monomers assemble into ordered fibrous arrays called fibrin. • Fibrin has a periodic structure that repeats every 23nm.

45. βDomain is specific for sequences of the form gly-his-arg. • γDomain binds gly-pro-arg. • The knob of the αsubunits fit into the holes on the γsubunits of another monomer to form a protofibiril, which is extended when the knobs of the b subunits fit into the holes of b subunits of other protofibrils.

46. Prothrombin is readied for activation by a vitamin K-dependent modification • Thrombin is synthesized as a zymogen called prothrombin. • Inactive form: 4 major domains. ① ② • Activation is begun by proteolytic cleavage of the bond between arg274 and thr275 • Cleavage of the bond between arg323 and ile324 yield active thrombin.

47. ※ Vitamin K (deficiency in this vitamin results in defective blood koagluation) • Essential for the synthesis of prothrombin and other clotting factor. • ※ Dicoumarol : vitamin K mimic antagonist. • - Binding of discoumarol results in synthesize an abnormal prothrombin that does not bind Ca2+(cofactor). Strange? Why? • Amino acid analysis  same A.A sequence Why? • NMR!

48. Normal prothrombin is carboxylated to γ–carboxyglutamate by a vitamin K-dependent enzyme. • γ–carboxyglutamate • can interact with Ca2+,which anchor • prothrombin phospholipid membrane. • -brings the zymogen into close • proximity to 2 clotting proteins converting into thrombin • -During activation, calcium-binding • domain is removed, freeing the thrombin • from the membrane so that it can • cleave fibrinogen and other targets. γ–carboxyglutamate

49. Hemophilia revealed an early step in clotting • -Hemophilia : the best known clotting defect. • sex-linked recessive characteristic. • In classic hemophilia, factor VIII of the intrinsic pathway is missing or has markedly reduced activity. • factor VIII is not a protease but it stimulate the activation of factor X by factor Ixa • Recombinant factor VIII • The activity of factor VIII is markedly increased by limited proteolysis by thrombin  positive feed back

50. The clotting process must be precisely regulated • Clots must form rapidly. • Activated clotting factors are short-lived because they diluted by blood flow, removed by the liver, and degraded by proteases. • Factor V and VIII are digested by protein C, switched on by the action of thrombin • Thrombin has a dual function: catalyzes the formation of fibrin and it initiates the deactivation of the clotting cascade