lecture10_10_7_08 - Lecture 10 Chapter 10 Regulatory...

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Unformatted text preview: Lecture 10 Chapter 10: Regulatory Strategies Overview: allosteric control (aspartate transcarbamoylase); isozymes; reversible covalent modification (protein kinases and phosphatases); proteolytic activation (chymotrypsin, trypsin, pepsin); transcriptional regulation DNA building blocks HW: 10.1, 10.5, 10.12, 10.18, 10.19 Aspartate transcarbamoylase (ATCase): allosteric inhibition Aspartate transcarbamoylase (ATCase): allosteric inhibition ATCase catalyzes the first step of pyrimidine biosynthesis; regulated by amount of CTP (end product) in the cell CTP, the end product, inhibits ATCase; feedback inhibition ATCase displays sigmoidal kinetics ATCase ATCase consists of two separable subunits: regulatory (r) and catalytic (c) The two subunits can be separated by reacting crucial cysteine residues with a mercury compound 2 c 3 + 3 r 2 -> c 6 r 6 Structure of ATCase The two subunits can be separated by using ultracentrifugation two catalytic trimers stack on top of one another PALA: a bisubstrate analog Active site of ATCase with bound PALA What are subunit roles? PALA, an analog of the two substrates, resembles an intermediate in the catalytic pathway T-to-R state transition in ATCase recall Hb structural changes and cooperative effects of oxygen binding ATCase kinetics concerted vs. sequential models: recall Hb high KM (T state) vs. low KM (R state) CTP stabilizes the T state compact, relatively inactive state (tense) expanded, relaxed state stabilized by PALA The R- and T-state equilibrium Effects of CTP versus ATP on ATCase kinetics CTP stabilizes the T state whereas ATP stabilizes the R state: heterotropic effects (effects of nonsubstrate molecules on allosteric enzymes) Isozymes: fine-tune metabolism during stages of development isozymes of LDH isoenzymes, or isozymes, differ in amino-acid sequence yet catalyze the same reaction; generally display different kinetic parameters (e.g., KM) or respond to different regulatory molecules The rat heart LDH, lactate dehydrogenase, isozyme profile changes in the course of development. ! H isozyme; " M isozyme; LDH-1 = H4; LDH-2 = H3M; LDH-3 = H2M2 ; LDH-4 = M3H; LDH-5 = M4 • Most vertebrates contain at least two genes for lactate dehydrogenase enzymes that make similar but non-identical polypeptides (75% identical) called M and H. • In embryonic tissue, both genes are equally active, resulting in equimolar amounts of the two gene products and statistical array of tetramers: M4, M3H, M2H2, H3M, and H4 in the ratios 1:4:6:4:1. These isozymes can be detected by differing electrophoretic mobilities. • As embryonic tissue multiplies and differentiates, the relative amounts of M and H change. • In pure heart tissue, H4 predominates. In skeletal muscle, which functions anaerobically under stress, M4 predominates. Isozymes: sign of tissue damage • Pyruvate (an important metabolic intermediate) can form a covalent complex with NAD+ in the LDH active site, suggesting a regulatory role. • Active muscle is anaerobic and produces a fair amount of pyruvate. High levels of pyruvate allosterically inhibit H4 but not M4. Covalent modification as a means of regulating enzyme activity Relationship to heart disease & clinical diagnostics: • Injury to cells in tissues with high levels of LDH causes release of LDH into bloodstream. LDH-1 (H4) is found in the heart. • During a heart attack (myocardial infarction), the serum levels of enzymes normally confined to the heart muscle begin to rise. • During lung injury or disease; increase in LDH-2 Normal levels: LDH-1 17–27% LDH-2 27–37% LDH-3 18–25% LDH-4 8–16% LDH-5 6–16% Patient with myocardial infarction: LDH-1 38% ‘LDH-1–LDH-2 flip’ LDH-2 32% LDH-3 16% LDH-4 8% LDH-5 6% modification can be reversible or irreversible Covalent modification as a means of regulating enzyme activity Protein kinase: one of the largest protein families; more than 500 homologous proteins in humans Phosphorylation as a means of regulating enzyme activity Serine and threonine are modified by one class of protein kinases and tyrosine residues by another. Tyrosine kinases, which are unique to multicellular organisms, play pivotal roles in growth regulation, and mutations in these enzymes are commonly observed in cancer cells. Phosphorylation is used to regulate many eukaryotic proteins; as much as 30% modified Phosphorylation as a means of regulating enzyme activity Phosphorylation as a means of regulating enzyme activity Why is phosphorylation such an effective means of controlling protein activity? • add two negative charges to protein; alter specific electrostatic interactions; alter substrate binding and catalytic activity of an enzyme • phosphoryl group can form three or more hydrogen bonds; tetrahedral geometry makes these bonds highly directional allowing for specific interactions with hydrogen-bond donors • free energy of phosphorylation is large (–12 kcal/mol provided by ATP); affect conformational equilibrium between different functional states (order of 104) • phosphorylation/dephosphorylation can occur over sec – hours; respond to physiological processes • many proteins/enzymes/substrates can be modified rapidly • ATP is an important phosphoryl donor (provides cellular energy); thus, links energy status of the cell to the regulation processes in metabolism • reversal of kinase effects: protein phosphatases • turn off signaling pathways • PP2A suppresses cancerpromoting activity of certain kinases • phosphorlyation and dephosphorylation are not simply the reverse of one another; why? Role of cyclic AMP (cAMP) cAMP: important intracellular messenger; hormone epinephrine (adrenaline) signals cyclization of ATP; cAMP activates protein kinase A (PKA); PKA phosphorylates and regulates key target proteins R = regulatory C = catalytic R chain: Arg-Arg-Gly-Ala-Ile Phosphorylation site of target proteins: Arg-Arg-Gly-Ser-Ile R chain is a pseudosubstrate that binds to C and keeps it from acting on substrates. The inhibitor shown here contains the pseudosubstrate sequence. C allosteric regulation and phosphorylation Binding of pseudosubstrate to protein kinase A Inhibitor makes multiple contacts: salt bridges with Arg and Glu; hydrophobic interactions with Ile and Leu Catalytic core: a deep cleft and residues 40–280 are highly conserved in essentially all protein kinases Protein kinase inhibitors: treatment of cancer, diabetes, inflammation, and a number of other diseases. The human genome encodes >500 protein kinases that share a catalytic domain conserved in sequence and structure, but which are notably different in how their catalysis is regulated. The development of inhibitors is possible, in which they regulate important cellsignaling pathways. Fig. 1. The structure of the catalytic domain of cAbl in complex with Gleevec M. E. M. Noble et al., Science 303, 1800 -1805 (2004) Fig. 3. Representative examples of the action of families of clinically tested inhibitors of protein kinase signaling M. E. M. Noble et al., Science 303, 1800 -1805 (2004) Fig. 2. Chemical structures of selected kinase inhibitors that are in the clinic or in clinical trials M. E. M. Noble et al., Science 303, 1800 -1805 (2004) A third type of enzyme regulation: zymogens Some enzymes acquire full activity as they spontaneously fold into their characteristic three-dimensional structures. Others are inactive until activated by cleavage of one or more specific peptide bonds. The precursor is called a zymogen or proenzyme. ATP (energy) not required, so this can occur outside of the cell (in comparison to phosphorylation) Examples of zymogens • digestive enzymes: hydrolyze proteins; synthesized as zymogens in stomach and pancreas • blood clotting: mediated by cascade of proteolytic activations in response to trauma • protein hormones: proinsulin is a peptide precursor of insulin • collagen: this fibrous protein of the skin and bones is derived from procollagen, a soluble precursor • developmental processes are controlled by activation of zymogens • programmed cell death (apoptosis): mediated by caspases, which are derived from procaspases; activated by various cellular signals Chymotrypsin: a digestive enzyme that hydrolyzes proteins in the small intestine. It’s precursor is chymotrypsinogen, which is synthesized in the pancreas and stored in granules. Upon signaling (hormone, nerve impulse), the granule contents are released into an appropriate duct, the duodenum. The three chains are linked by two interchain disulfide bonds. Overlay of chymotrypsinogen and chymotrypsin: note movement of Ile 16 and important contact with its aminoterminus and Asp 194. *Cleavage occurs between amino acids 15 and 16. Electrostatic interaction triggers other movements and creation of active site. Generation of trypsin from trypsinogen: activation of other zymogens Specific inhibitors of proteolytic enzymes What is the significance of very low Kd? pancreatic trypsin inhibitor: • 6 kDa • binds tightly to active site (estimated Kd = 0.1 pM; standard free energy -18 kcal/mol) • needs to be denatured before it will dissociate • energy derived from numerous hydrogen bonds and salt bridge • free and bound inhibitor structures almost identical (preorganized structure) Other protease inhibitors • !1-antitrypsin: protects tissues from digestion by elastase mutations in antitrypsin can lead to ineffective inhibition or slower secretion from liver cell; elastase can then destroy tissue and fiber in the lungs leading to emphysema (destructive lung disease) • cigarette smoke can lead to oxidation of methionine 358 of the inhibitor, a residue essential for elastase binding Other protease inhibitors • antithrombin III: controls blood clotting • blood clots are formed by a cascade of zymogen activations • rapid response to trauma • release of tissue factor (an integral membrane glycoprotein) during trauma triggers cascade • thrombin is the key enzyme in clotting • thrombin signals other enzymes in cascade (positive feedback) Structure of fibrinogen two triple-stranded !-helical coiled coils & globular domains at ends 340 kDa; six chains; two each of A!, B", and # four Arg-Gly bonds are hydrolyzed cleavage produces fibrinopeptides A (18 aa) and B (20 aa) Role of fibrin intermediate Loss of the fibrinopeptides A and B from fibrinogen leaves a fibrin monomer subunit called !"# Fibrin monomers assemble spontaneously into fibrous arrays called fibrin Notice repeat every 23 nm Formation of fibrin clot globular domains at C termini of " and # chains have ‘holes’ that interact with peptides (" binds H3N+-Gly-His-Arg and # binds H3N+-Gly-Pro-Arg sequences) that are exposed at the N termini of " and # chains upon thrombin cleavage (referred to as ‘knobs’) B A " # protofibril Transglutaminase (factor XIIIa) catalyzes cross-link formation Factor XIIIa is produced by thrombin cleavage of protransglutaminase Prothrombin -> thrombin Vit K important for prothrombin synthesis phylloquinone phytyl ‘soft clot’ is stabilized by formation of amide bonds between side chains of Lys and Gln found in Gla Vit K antagonists from spoiled sweet clover (also in rat poison) O The role of vitamin K ! H N O The role of vitamin K N H – N H HR HS CO2 " O ! HR H N O Dihydrovita min K O2 O H CH3 O R O H HS C COOH OOC COO– " COO– Glu O2 H2O Gla An NADPH-dependent reductase reduces the vitamin K quinone to its hydroquinone form. Conversion of Glu to Gla requires the reduced vitamin K as well as O2 and CO2. Glu s ide c hain Dihydrovita min K OH CH3 Vitamin K-2R ,3S-epoxide O CH3 O phytyl phytyl O H O peroxide epoxide O C O H2O H – OH C COOH Glu s ide c hain Vit K is essential for formation of !-carboxyglutamate (Gla)containing proteins, which are needed in blood clotting A peroxide intermediate is proposed to be involved in the mechanism. Role of vitamin K and calcium A vitamin K-dependent carboxylation reaction converts glutamate into !carboxyglutamate, a strong chelator of calcium (Ca2+). Prothrombin binds strongly to calcium. This helps to anchor the zymogen to phospholipid membranes derived from blood platelets after injury. This process brings prothrombin in close proximity to two clotting proteins that catalyze its conversion to thrombin. Hemophilia: clotting defect • factor VIII is missing or has reduced activity • factor VIII stimulates activation of factor X, the final protease of the pathway, by factor IXa, a serine protease ...
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This note was uploaded on 08/04/2010 for the course CHM 6620 taught by Professor Dr.christinechow during the Fall '08 term at Wayne State University.

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