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Protein Structure and Function

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  1. Protein Structure and Function

  2. CHAPTER2. From Structure to Function

  3. There are many levels of protein function • Biochemist: biochemical role of an individual protein • Cell biologist, geneticist: cellular roles judged by the phenotype of its deletion or signal pathway • Physiologist, developmental biologist: cellular or organism level • Protein can have many functions on different levels  ex) tubulin • 4 fundamental biochemical functions: Binding, catalysis, switching and structural elements

  4. 2-0. Overview : The Structural Basis of Protein Function ◈ Protein interaction and function Figure 2-1. The functions of tubulin Ex) GTPase(Protein switch) depend on both binding and catalytic function.

  5. 2-0. Overview : The Structural Basis of Protein Function Radial spoke Assemblies of microtubules, dynein and other microtubule-associated proteins form flagella that propel sperm Microtubules and associated motor proteins form a network of “tracks” on which vesicles are moved around in cells. Taxol anti-cancer drug Figure 2-1. The functions of tubulin

  6. TAXOL • 미국 주목나무의 주피(bark)에서 추출한 taxane ring을 가진 alkaloid이다. Taxol 와 a-, b- tubulin complex 구조 • Mechanism of ActionPaclitaxel은 1992년12월에 난치성 난소암의 치료제로 FDA의 승인을 받은 항암제로 세포 내 microtubule의 assembly를 증진시키고 disassembly를 저해함으로써 항암효과를 나타내는 독특한 약물이다 • Paclitaxel은 암세포의 microtubule의 assembly를 증진 시키고 일단 형성된 microtubule을 안정화시켜 polymerization 상태로 남아있게 함. microtubule의 disassembly를 저해하여 유사분열에 필요한 방추사의 형성을 억제하므로 세포 주기상 암 세포가 G2기와 M기에 머무르게 되어 cytotoxic effect를 가진다. 이 세포주기는는 방사선에 매우 민감한 주기이므로 radiation therapy와 병용할 시 cytotoxicity가 증가하게 됨.

  7. 2-1. Recognition, Complementarity and Active Sites ◈ Ligand binding - Ligand or substrate binding is very specific. - Specificity arises from the complementarity of shape and charge distribution between the ligand and its binding site on the protein surface Figure 2-2. Substrate binding to anthrax toxin lethal factor

  8. 2-1. Recognition, Complementarity and Active Sites • Molecular recognition depends on specialized microenvironments that result from protein tertiary structure; • Internal cavities, pocket or cleft of surface • Specialized microenvironments at binding sites contribute to catalysis; • Lysine in hydrophobic env  as acid • Enzyme favor reaction by providing a nonpolar environment ◈ Substrate binding • The proximity of these two (Lys164 and Lys166) positive charges lowers the proton affinity of both of them, making lysine 166 a better proton shuttle for the metal-bound substrate. Figure 2-3. Schematic of the active site of mandelate racemase showing substrate bound

  9. 2-2. Flexibility and Protein Function ◈ Ligand binding model • The flexibility of tertiary structure allows proteins to adapt to their ligands; • Charge configuration, potential H-bond complementarity • Lack-and-key analogy : implies rigidity of the protein(the lock) and of the ligand(the key) • Induced fit model : both protein and the ligands are naturally flexible Figure 2-4. Tight fit between a protein and its ligand

  10. 2-2. Flexibility and Protein Function ◈ Protein structure flexibility Peptide analog of the natural substrate Haloperidol Crixivan Each inhibitor clearly has a quite different structure, yet all bind tightly to the active site(Because of protein structure flexibility) Figure 2-5. HIV protease, and enzyme from the virus that causes AIDS, bound to three different inhibitors

  11. 2-2. Flexibility and Protein Function • Protein flexibility is essential for biochemical function • Red : increase in enzyme activity from yeast • Blue : increase in enzyme activity from Thermophilic bacteria(optimal growth rate is at 85℃) • Balance between flexibility and rigidity is necessary for protein activity because stability has been achieved at the expense of flexibility. • Genetic engineering in Enzyme? Figure 2-6. Differences in the temperature dependence of the specific activity of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from two organisms

  12. 2-2. Flexibility and Protein Function Some proteins undergo very large shape changes when the correct ligand binds Figure 2-7. Example of a large conformational change in adenylate kinase

  13. 2-3. Location of Binding Sites ◈ Binding sites (concave, convex, or flat) Growth hormone Receptors Two different protein-protein interfaces can be made by one molecule of growth hormone with two identical receptor molecules Figure 2-8. The complex between human growth hormone and two molecules of its receptor

  14. 2-3. Location of Binding Sites Helix-turn-helix motif Zinc finger Many binding sites for RNA or DNA on proteins are protruding loops or alpha helices that fit into the major and the minor grooves of the nucleic acid Figure 2-9. Two protein-DNA complexes

  15. 2-3. Location of Binding Sites • Binding sites for small ligands are clefts, pockets or internal cavities because it provides microenvironment to block water access, which is important for many enzyme reactions and enough contact points to bind strongly. • The active site of this enzyme contains a catalytic heme group(puple) most of which is completely buried inside the protein. • protein flexibility open a transient path for • Penetration of the substrate Figure 2-10. Structure of bacterial cytochrom P450 with its substrate camphor bound

  16. Cytochrome P450 • The cytochrome P450 superfamily (officially abbreviated as CYP) is a large and diverse group of enzymes. The function of most CYP enzymes is to catalyze the oxidation of organic substances. The substrates of CYP enzymes include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for about 75% of the total number of different metabolic reactions.[1] • The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: • RH + O2 + NADPH + H+ → ROH + H2O + NADP+ • Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a heme cofactor and, therefore, are hemoproteins. CYPs use a variety of small and large molecules as substrates in enzymatic reactions. Often, they form part of multi-component electron transfer chains, called P450-containing systems. Cytochromes P450 have been named on the basis of their cellular (cyto) location and spectrophotometric characteristics (chrome): when the reduced heme iron forms an adduct with CO, P450 enzymes absorb light at wavelengths near 450 nm, identifiable as a characteristic Soret peak. • CYP enzymes have been identified in all domains of life, i.e., in animals, plants, fungi, protists, bacteria, archaea, and even viruses.[2][3] More than 11,500 distinct CYP proteins are known.[4]

  17. 2-3. Location of Binding Sites • Catalytic sites often occur at domain and subunit interfaces • If a protein has more than one structural domain, then the catalytic site can be found at the interface between them Figure 2-11. Structure of the dimeric bacterial enzyme 3-isopropylmalate dehydrogenase

  18. 2-4. Nature of Binding Sites ◈ Binding site characteristic • Binding sites generally have a higher than average amount of exposed hydrophobic surface Heme Large hydrophobic areas on the surface of a protein lead to self-association And oligomerization. But binding sites for small molecules are usually too small and concave to allow the protein to oligomerize. Figure 2-12. Surface view of the heme-binding pocket of cytochrome c6, with hydrophobic residues indicated in yellow

  19. 2-4. Nature of Binding Sites ◈ Week binding and partner swapping • Weak interactions can lead to an easy exchange of partners • Weak interaction → easy to dissociation and combine with other protein partner → PARTNER SWAPPING • STAT molecules are themselves phosphorylated, and their SH2 domains dissociate from the receptor and bind to the phosphotyrosine on the other STAT molecule to form an active signaling dimer. This results in two SH2-p-Tyr interactions instead of one SH2-p-Tyr interaction with receptor. It should Irreversible for signal transduction Figure 2-13. Partner swapping in a signaling pathway

  20. 2-4. Nature of Binding Sites ◈ Week binding and domain swapping • Domain swapping is also mediated by week interaction between domains • Conformational rearrangement of the carboxy-terminal arm of the papillomavirus capsid protein required for the stable assembly of the virus coat • PAK1 protein kinase is a domain-swapped dimer • A regulatory domain from each monomer inhibit • the active site of the other monomer. • When an activator protein (GTP-bound Cdc42) binds to this regulatory domain • It relieves the domain swapping and frees the active site. Figure 2-14. Domain swapping in the papilloma virus capsid protein

  21. Displacement of water also drives binding events • for binding between ligand and protein, water layer (hydrogen shell) must be disrupted or partially displaced. • Thermodynamics? • Enthalphy? Between Waters and protein-waters, in hydrophobic environment • How about entrophy?

  22. 2-4. Nature of Binding Sites ◈ Interaction between oleate and lipid-transport protein nsLTP • Hydrogen bond to the charged head group • Mainly hydrophobic interaction with the lipid tail Generally, the affinity between a protein and its ligand is due to hydrophobic interactions,which are non-directional, whereas specificity of binding is chiefly due to directional forces such as hydrogen bonding. Lipid oleate Figure 2-15. Ligand binding involving hydrophobic and hydrogen-bond interactions

  23. 2-5. Functional Properties of Structural Proteins ◈ Proteins as frameworks, connectors and scaffolds • In some cases, structural proteins are assisted by DNA and RNA, lipid and carbohydrate molecules. • Ex). Ribosome: has over a hundred different protein components(for stabilization, catalytic function) • Dynamics in proteins; • muscle, actin filaments, fibrinogen Blue : protein Red & grey : RNA Figure 2-16. Structure of the 50S (large) subunit of the bacterial ribosome


  25. 2-5. Functional Properties of Structural Proteins ◈ Some structural proteins only form stable assemblies • Permanent structural proteins • Silk, collagen, elastin, or keratin • Collagen : fibrous component of tendons. • Collagen is a triple helix of three protein chain made up repeating GlyXY sequence(where X is often proline). • - Hydrophobic interactions, H-bond and cross-linking of lysine residues (peptidyl aldehydes by lysyl oxidase) Figure 2-17. Structure of collagen

  26. 2-5. Functional Properties of Structural Proteins ◈ Some structural proteins serve as scaffolds • In the signal transduction, specific protein recruitment is required. • Sometimes this recruitment occurs by localizing components to the cell membrane. • But the other cases specific structural proteins serve as scaffold proteins Figure 2-18. The Ste5p scaffold in MAPK cascade

  27. 2-6. Catalysis: Overview ◈ Catalysis and Catalyst • Catalysts accelerate the rate of a chemical reaction without changing its overall equilibrium • Catalyst : substance that accelerates the rate of a chemical reaction without itself becoming permanently altered in the process • orotidine 5’-monophosphate decarboxylase reaction does not occur readily at room temperature in the absence of a catalyst. Figure 2-19. The enzyme orotidine 5’-monophosphate decarboxylase catalyzes the transformation of orotidine 5’-monophosphate to uridine 5’-monophosphate

  28. 2-6. Catalysis: Overview ◈ Catalysis and Catalyst • OMP decarboxylase is not the only enzyme that is known to accelerate a reaction by more than a billion fold. Figure 2-20. Table of the uncatalyzed and catalyzed rates for some representative enzymatic reactions

  29. 2-6. Catalysis: Overview ◈ Catalysis is reducing the activation-energy barrier to a reaction • - Blue : Uncatalyzed reaction. • single transition-state barrier • Red : In presence of CATALYST. • additional smaller activation-energy barriers • -The energy required to overcome this barrier is known as the activation energy • :activation-energy barrier • The higher activation energy barrier, the slower the reaction • How to overcome the barrier? S P Figure 2-21. Energetics of catalysis

  30. 2-7. Active-Site Geometry • Reactive groups in enzyme active sites are optimally positioned to interact • With the substrate • -primarily by van der Waals interactions and complementary H-bonds and electrostatic interactions • -Too tight substrate binding reduces efficiency as a catalyst. Why? • -Specific complex is necessary for productive collision; correct orientation • Which induces atomic orbitals can overlap to allow the appropriate bonds to be formed or broken. • -enzyme offer the time and place for second substrate to bind to enzyme. Negative substrate Gated binding Positive potential Figure 2-23. Schematic diagram showing some of the ways in which electrostatic interactions can influence the binding of a ligand to a protein

  31. 2-7. Active-Site Geometry ◈ Active site residues are optimally positioned to interact with the substrate Active site • This enzyme is shown as a homodimer and two active site • Negative potential on the protein will repel the negatively charged superoxide substrate(O2-) • Positive potential in the active site will attract it. Active site Red contour : negative electrostatic potential Blue contour : positive electrostatic potential Figure 2-22. The electrostatic potential around the enzyme Cu, Zn-superoxide dismutase

  32. 2-7. Active-Site Geometry ◈ Specificity sub-site and Reaction sub-site • Reaction sub-site : enzyme carry out the chemistry • Specificity sub-site : enzyme uses polar and nonpolar groups to make weak interactions with the substrate. • A.A change in this sites for medical and industrial uses PLP Figure2-24. Schematic diagram of the active site of E.coli aspartate aminotransferase

  33. 2-8. Proximity and Ground-State Destabilization ◈ Some active sites chiefly promote Proximity • Proximity factor : substrate molecules are intrinsically reactive. Close to each other • so that the atomic orbitals are positioned to overlap for the new chemical bond. • Concentration? Close together Figure2-25. Catalysis of the reaction of carbamoyl phosphate and aspartate by the enzyme aspartate transcarbamoylase depends on holding the substrates in close proximity and correct orientation in the active site

  34. 2-8. Some active sites destabilize Ground-State Destabilization • Chorismate mutase for the biosynthesis of aromatic amino acid destabilize ground states to increase the reaction rate. • Enzyme promotes the reaction primarily by binding the substrate in an unusual “CHAIR” conformation • Compounds designed to mimic this conformation are particularly effective inhibitors of the enzyme. Negative charged carboxylate group in hydrophobic pocket Energetically unfavorable Figure2-26. The pericyclic rearrangement of chorismate to prephenate via the proposed “chair-like” transition state

  35. 2-9. Stabilization of Transition States and Exclusion of Water ◈ Binding energy If the transition state can be bound more tightly than the substrate, activation energy will be reduced The differential binding of enzyme for these two state Is the driving force of reactions a = new interactions with transition state b = stabilization ES : enzyme and substrate complex EP : enzyme and product complex Substrate and product are of equal energy, with a large free-energy barrier between them. Figure2-27. Effect of binding energy on enzyme catalysis

  36. Many active sites must protect their substrates from water, but must be accessible at the same time • When an active site is in its closed conformation, it is protected from water. But substrates, products?

  37. 2-9. Stabilization of Transition States Hydrogen bond Acetyl group addition Attack • Citrate synthase with a different geometry from that of the substrate • Asp375 and His274 catalyze the formation of the enol of acetyl-CoA • The acetyl-CoA enol attacks the carbonyl carbon of oxaloacetate • Addition of the elements of the acetyl group at this portion Figure2-28. The active site of citrate synthase stabilizes a transition state with a different geometry from that of the substrate

  38. 2-9. Stabilization of Transition States and Exclusion of Water ◈ Binding and conformational changes - If an enzyme active site dies not start out perfectly complementary to the transition state, the enzyme undergo conformational changes that increase that complementarity Complex with its substrate Unliganded PGK Figure2-29. Phosphoglycerate kinase (PGK) undergoes a conformational change in its active site after substrate binds

  39. 2-9. Stabilization of Transition States and Exclusion of Water • Many active sites must protect their substrates from water, • but must be accessible at the same time • Resting enzyme exist in an open state to which substrates can bind readily • Substrate binding trigger the conformational changes to the closed form • Opening and/or closing of the lid is the rate-determining step in the reaction Hydride ions (H-) are unstable in water, so the active site must be shield from bulk solvent. Why? Figure2-30. NAD-dependent lactate dehydrogenase has a mechanism for excluding water from the active site once substrates are bound

  40. 2-10. Redox Reactions ◈ Enzyme chemical reactions A mammalian cell produces over 10,000 different proteins → more than half are enzymes → thousands of different enzymes with thousands of different substrates and products → 4 similar reaction → Oxidation/reduction → addition/elimination → hydrolysis → decarboxylatoin

  41. 2-10. Redox Reactions 1). Oxidation and reduction reaction → The oxidation of an alcohol to a ketone by NAD as in the reaction catalyzed by malate dehydrogenase → The oxidation of a satuated carbon-carbon bond to an unsaturated carbon-carbon bond by FAD as in the reaction catalyzed by succinate dehydrogenase Figure2-31. Example of oxidation/reduction reacions

  42. 2-10. Redox Reactions 1). Oxidation and reduction reaction → In the first step of the pathway for the conversion of cholesterol to pregnenolone Oxidation by insertion of an oxygen atom Figure2-31. Example of oxidation/reduction reacions

  43. 2-11. Addition/Elimination, Hydrolysis and Decarboxylation 2). Addition and elimination reaction → Addition of water across the C=C of fumarate to create the HC-COH group of malate, a reaction catalyzed by fumarase. Only L-malate → Addition of acetate to the carbonyl carbon of oxaloacetate in the aldol condensation reaction catalyzed by citrate synthase Figure2-32. Examples of addition/elimination reactions

  44. Ordered binding; Oxaloacetate binding induces a major conformational change Leading to the creation of a binding site for acetyl CoA

  45. 2-11. Addition/Elimination, Hydrolysis and Decarboxylation 3). Hydrolysis reaction → Cleavage of the C-N bond of a peptide involves attack by water on the carbonyl carbon, resulting in formation of a caboxylic acid and an amine (by Protease) → Breaking the P-O-R bond of a phosphate diester involves attack by water on the phosphorus atom, resulting in formation of a phosphate monoester and an alcohol (by endonuclease) Figure2-33. Examples of peptide and phosphoester hydrolysis

  46. Tetrahedral intermediate (acyl-enzyme) Serine protease mechanism Nucleophilic attack Amine is free Substrate binding Water mediated deacylation Carboxylic acid product OH- Attacks the carbonyl carbon

  47. 2-11. Addition/Elimination, Hydrolysis and Decarboxylation 4). Decarboxylation reaction → Shortening of the three-carbon unit of pyruvate to the two-carbon unit of acetaldehyde is accomplished by the loss of CO2, catalyzed by the cofactor TPP bound at the active site of the enzyme pyruvate decarboxylase Figure2-34. Example of the decarboxylation of a carboxylic acid

  48. Pyruvate Dehydrogenase Links Glycolysis to the Citric acid cycle

  49. 1. Decarboxylation

  50. 2. Oxidation 3. Formation of Acetyl CoA