9.1 • Specificity Is the Result of Molecular Recognition

1. 9

2. 9.1 • Specificity Is the Result of Molecular Recognition “Induced Fit” and the Transition-State Intermediate

4. Key and lock Induce fit

5. The induced conformational change in hexokinase M11.8 Induced fit is the main model for changes in the enzyme to fit the transition state and to bring functional side groups of the enzyme to the right location to help the catalysis.

6. 8.2 • Controls Over Enzymatic Activity— General Considerations

7. Regulatory strategies • Product accumulates(The enzymatic rate “slows down” as product accumulates and equilibrium is approached); • Substrates and cofactors(The availability of substrates and cofactors will determine the enzymatic reaction rate); • Allosteric control(aspartate transcarbamoylase, hemoglobin); • Multiple forms of enzymes(lactate dehydrogenase); • Reversible covalent modification(e.g. phosphorylation/dephosphorylation, protein kinase A);

8. Proteolytic activation(proteases involved in digestion, blood clotting). • Regulation at protein level often allows a faster response to changes in cellular conditions (substrate concentrations, hormone action, etc.) than regulation of gene expression. • Changes of 3D-structures of proteins are pivotal in many mechanisms of regulation at protein level. • Changes in structure regulate binding of substrates (e.g. formation of active sites in enzymes) or interactions with other proteins (e.g. covalent modifications like phosphorylation in signal transduction).

9. Specialized controls: Enzyme regulation is an important matter to cells, and evolution has provided a variety of additional options, including zymogens, isozymes, and modulator proteins

10. zymogens Most proteins become fully active as their synthesis is completed and they spontaneously fold into their native, three-dimensional conformations. Some proteins, however, are synthesized as inactive precursors, called zymogens or proenzymes, that only acquire full activity upon specific proteolytic cleavage of one or several of their peptide bonds.

11. Proteolytic activation • Many proteins/enzymes are made as inactive precursors that are activated by proteolytic cleavage: after this cleavage the enzymes fold into their catalytically active conformation. • Inactive precursors are called proenzymes or zymogens. • Proteolytic activation is irreversible -> a different mechanism is required for inactivation of the enzyme. • Examples: blood clotting, digestive proteases, peptide hormones (insulin), proteins involved in developmental processes (collagen), apoptosis (programmed cell death; caspases) and virus proteases (cuts multidomain viral proteins into their active forms, see 9.1.7).

12. Example 1: digestive proteases Proteases involved in digestion of proteins in the stomach and small intestine are synthesized as inactive precursors (pancreas). They are activated by proteolytic digestion after secretion into the lumen of the intestine.

13. Chymotrypsin. • Made as an inactive precursor (chymotrypsinogen) in the pancreas. • Activation by cleavage of the peptide bond between Arg-15 and Ile-16 with trypsin -> -chymotrypsin; cleavage results in a small conformational change with large consequences ! • -Chymotrypsin removes 2 dipeptides (residues 14-15 and 147-148) from other -chymotrypsin molecules -> -chymotrypsin. Activity does not change after this second proteolytic step: -chymotrypsin is already fully active.

14. Insulin. Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin, an important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin

15. Isozymes LDH (lactate dehydrogenase) isozymes differ in their allosteric regulation by pyruvate.

16. The kinetic properties of the various LDH isozymes differ in terms of their relative affinities for the various substrates and their sensitivity to inhibition by product. Different tissues express different isozyme forms, as appropriate to their particular metabolic needs. By regulating the relative amounts of A and B subunits they synthesize, the cells of various tissues control which isozymic forms are likely to assemble, and, thus, which kinetic parameters prevail.

17. Example 2: Blood clotting Blood clotting results from a cascade of reactions. In a cascade, a signal initiates a series of steps, each of them catalyzed by an enzyme. At each step the signal is amplified. In blood clotting the activated form of one clotting factor catalyzes activation of the next. Very small amounts of the initial factors trigger the cascade, => rapid response to trauma (e.g., damage to a blood vessel).

18. Blood clotting Fibrin fiber

19. Conversion of fibrinogen to fibrin causes clotting. The final step of clotting is conversion of fibrinogen to fibrin by thrombin, a protease. Fibrinogen has 6 protein chains (2x A, B and ), folded into globular units connected by rods. Thrombin cleaves 4 peptides from the A and B chains in the central globule, resulting in fibrin monomer ()2.

20. Carboxyl ends of the - and  chains interact with the newly exposed N-terminal regions => polymerization (protofibrils).

21. Fibrils are stabilized by cross-linking: formation of amide bonds between lysine and glutamine by transglutaminase, which is activated from protransglutaminase by thrombin. The network of fibrils forms the clot.

22. Activation of thrombin. Thrombin activates fibrinogen, but how is thrombin activated ? Thrombin is activated by proteolytic activation of prothrombin with factors Xa (also a protease) and Va. Activation removes a gla and 2 kringle domains. Modular structure of prothrombin

23. Modulator Proteins inactive Modulator proteins are proteins that bind to enzymes, and by binding, influence the activity of the enzyme. active Cyclic AMp- dependent protein kinase is a 150- to 170-kD R2C2 tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10-8M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits are enzymatically active as monomers

24. Covalent modification (Reversible) covalent modification alters properties of proteins (for example: enzyme activity).

25. Some modifications are reversible (e.g. phosphorylation by kinases dephosphorylation by phosphatases). • Others are irreversible: • signal-transduction proteins like Ras and Src are attached to the plasma membrane by irreversible attachment of a lipid • irreversible attachment of ubiquitin to a protein is a signal that the protein should be destroyed • proteolysis is always irreversible • All metabolic pathways are partially regulated by covalent modifications.

26. Many enzymes/proteins involved in signal transduction and membrane channels are regulated by phosphorylation by protein kinases. Protein kinases transfer the -phosphate group of ATP to the OH-side chains of specific serine or threonine residues or to tyrosine groups of a specific acceptor protein.

27. Only intracellular proteins are modified by phosphorylation with kinases. Phosphate groups can be removed from proteins by different enzymes: protein phosphatases.

28. Examples of cellular processes regulated by Ser/Thr protein kinases and the signals to which they respond.

29. Phosphorylation and dephosphorylation are not the reverse of each other: the net result is conversion of ATP into ADP + Pi (G = -12 kcal/mol). The rate of cycling depends on the relative activities of the kinases and phosphatases involved.

30. Phosphorylation of proteins: • changes electrostatic interactions by addition of two negative charges per phosphate; • may result in 3 H-bonds per phosphate; • changes the free energy of a protein with appr. -6 kcal/mol; this energy can be used for conformational changes; • is kinetically controlled (can be slow or fast, depending on the activity and amount of kinases and phosphatases); • can evoke amplified effects: a single kinase can phosphorylate hundreds of target proteins (e.g. protein kinase A); • is linked to the energy status of a cell (as ATP is the phosphate donor).

31. Protein kinases are a very large family of enzymes (559 homologues in human): • Dedicated protein kinases: phosphorylate single proteins or groups of related proteins • Multifunctional protein kinases: have very different target molecules • Many protein kinases recognize the target motif: • Arg - Arg - X - (Ser/Thr) - Z • (Ser or Thr being the site of phosphorylation, X a small residue and Z a large hydrophobic residue)

32. Example: protein kinase A Protein kinase A modulates the activity of many proteins by phosphorylation. Protein kinase A occurs in an inactive form (R2C2), consisting of two regulatory (inhibiting) subunits and two catalytic subunits, and in an active form (2C). cAMP activates protein kinase A by dissociation of the R-subunits from the R2C2-complex (allosteric activation). The R-subunits contain a pseudosubstrate sequence: Arg - Arg - Gly - Ala - Ile, which binds to the catalytic site of the C-subunits. Binding of cAMP allosterically moves the pseudosubstrate sequence out of the catalytic sites.

33. Protein kinases bind ATP and target protein in a cleft. • ATP and the inhibitor bind in a deep cleft between two lobes of protein kinase A: • one lobe binds ATP-Mg2+; • the other lobe binds the inhibitor and contains the catalytic residues; • the arginines of the inhibitor bind to carboxylates in PKA; the nonpolar Z-residue binds in a hydrophobic groove in PKA; • large part of this structure is conserved in other protein kinases.

34. 8.3• The Allosteric Regulation of Enzyme Activity

35. Allosteric control • Allosteric proteins/enzymes: • two or more interacting substrate binding sites (usually two or more subunits) • different conformations of the substrate binding sites, one with high affinity, another with low affinity for the substrate • separate sites for binding of effector molecules; effector molecules either increase or decrease the affinity for substrate (allosteric activators or inhibitors)

36. binding of substrate or effector molecules to one subunit influences binding of substrates to the other subunits in the same enzyme molecule (cooperativity): • positive cooperativity: binding of substrate or effector to one site increases affinity for substrate of the other subunits (R-state curve) • negative cooperativity: binding of effector decreases affinity for substrate of the other subunits (T-state curve) • allosteric enzymes are often found in the beginning of a metabolic route: their activity controls the pathway.

37. Example 1: Aspartate transcarbamoylase (ATCase). ATCase catalyzes the first step in synthesis of pyrimidines (CTP, UTP and TTP), precursors for DNA and RNA. The amounts of pyrimidines in a cell must balance the amounts of purines (ATP and GTP).

38. ATCase is inhibited by CTP, the final product of the pathway (feedback inhibition) and stimulated by ATP. CTP and ATP are structurally very different from the substrates and bind to a site distinct from the active site: allosteric inhibition/activation.

39. ATCase can be separated in two kinds of subunits by modification of cysteines (p-hydroxymercuribenzoate). Subunits can be separated by ion-exchange chromatography (charge) or utltracentrifugation (size). Ultracentrifugation of ATCase (A) and ATCase after modification of cysteines (B).

41. PALA {(N-phosphonacetyl)-L-acetate} is a bi-substrate analogue that resembles a transition state intermediate during catalysis: it binds in the active site; PALA binds at the boundary between two adjacent c-chains. Suitable amino acid residues are available for recognition of all features of PALA (charges, H-bonds).

42. Binding of PALA causes a change in quaternary structure: • catalytic trimers move 12Å apart and rotate 10o; • regulatory dimers rotate 15o. • T (tense) state: low affinity for substrate; low catalytic activity • R (relaxed) state: high affinity for substrate; high activity • Equilibrium between T and R-forms is dependent on the number of substrate molecules bound to the enzyme.

43. CTP binds to the r-subunits far (50 Å) from the active site. CTP binding stabilizes the T-state.

44. The transition between T- and R-state affects all 6 catalytic sites of ATCase equally and at the same time (‘all or none’): concerted mechanism for allosteric regulation. Without substrates or regulators, the T-state is favored 200 times over the R-state (L = 200).

45. Allosteric enzymes: • Do not follow Michaelis-Menten kinetics, but have sigmoidal kinetics as a function of substrate concentration; • Are susceptible to regulation by other molecules (for example: end products of a metabolic route);

46. At low substrate concentration: enzyme in T-state with low affinity (Km) for substrate and low activity; • At higher substrate concentration: switch to R-state with high affinity for substrate and high activity. Binding of substrate to one site increases activity of other five sites (cooperativity); • Sigmoidal curve is composite of two Michaelis-Menten curves; • Isolated c-subunits have activity corresponding to the R-state curve.

47. In the presence of allosteric inhibitor (CTP): enzyme is less responsive to cooperative effects of substrate binding (increase of apparent Km). An allosteric activator (ATP) increases the reaction rate at a given substrate concentration (decrease of apparent Km). • High ATP stimulates ATCase: • to balance purine and pyrimidine pools; • because it indicates that sufficient energy is available for DNA and mRNA synthesis.

48. Influences of substrates on allosteric enzymes: homotropic effects. • Influence of non-substrate molecules on allosteric enzymes: heterotropic effects. • Other example: • phosphofructokinase: tetramer, control of glycolysis • Fructose-6P + ATP -> fructose 1,6-biP + ADP • allosteric inhibition by ATP, positive regulation by AMP

49. Models of allosteric control. • Concerted model: allosteric enzyme exists in two states, T and R; there are no intermediate states; • Sequential model: binding of one molecule of substrate increases the affinity of neighboring sites without a transition of the entire enzyme. • Many enzymes display a behavior that is a combination between sequential and concerted models (e.g. hemoglobin) • Concerted and sequential substrate binding models are described by different mathematical formulas. (Mathematical description of cooperativity (10.1.5) is part of Enzymology).

50. Sequential (top) and concerted (bottom) models of allosteric regulation.