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Transcript
Transport & Signaling; Nucleic Acid Chemistry Andy Howard Introductory Biochemistry 30 September 2008 Biochemistry:Transport; Nucleic Acids 09/30/08 What we’ll discuss Membrane transport Energetics Active transport Transporting big molecules Membrane Signaling Nucleic acid chemistry Adenylyl cyclase Inositol-phospholipid signaling pathway Receptor tyr kinases Pyrimidines: C, U, T Purines: A, G Nucleosides 09/30/08 Biochemistry:Transport; Nucleic Acids p. 2 of 50 Protein-facilitated passive transport All involve negative DGtransport Uniport: 1 solute across Symport: 2 solutes, same direction Antiport: 2 solutes, opposite directions Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow These proteins can be inhibited, reversibly or irreversibly 09/30/08 Biochemistry:Transport; Nucleic Acids Diagram courtesy Saint-Boniface U. p. 3 of 50 Kinetics of passive transport Michaelis-Menten saturation kinetics: v0 = Vmax[S]out/(Ktr + [S]out) … we’ll revisit this after we do enzyme kinetics Vmax is velocity achieved with fully saturated transporter Ktr is analogous to Michaelis constant: it’s the [S]out value for which half-maximal velocity is achieved. 09/30/08 Biochemistry:Transport; Nucleic Acids p. 4 of 50 Primary active transport Energy source: usually ATP or light Energy source directly contributes to overcoming concentration gradient QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Bacteriorhodopsin PDB 1F50, 1.7Å 25 kDa monomer Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production P-glycoprotein: ATP-driven active transport of many nasties out of the cell 09/30/08 Biochemistry:Transport; Nucleic Acids p. 5 of 50 Secondary active transport Active transport of one solute is coupled to passive transport of another Net energetics is (just barely) favorable Generally involves antiport QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Pyrococcus Multisugar transporter PDB 1VCI 83 kDa dimer Bacterial lactose influx driven by proton efflux Sodium gradient often used in animals 09/30/08 Biochemistry:Transport; Nucleic Acids p. 6 of 50 Complex case: Na+/K+ pump Typically [Kin] = 140mM, [Kout] = 5mM, [Nain] = 10 mM, [Naout] = 145mM. ATP-driven transporter: 3 Na+ out for 2 K+ in per molecule of ATP hydrolyzed 3Na out: 3*6.9 kJmol-1, 2K in: 2*8.6 kJmol-1 = 37.9 kJ mol-1 needed, ~ one ATP 09/30/08 Biochemistry:Transport; Nucleic Acids Diagram courtesy Steve Cook p. 7 of 50 What’s this used for? Sodium gets pumped back in in symport with glucose, driving uphill glucose transport That’s a separate passive transport protein called GluT1 to move glucose back 09/30/08 Biochemistry:Transport; Nucleic Acids Diagram courtesy Steve Cook p. 8 of 50 How do we transport big molecules? Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis Special types of lipid vesicles created for transport 09/30/08 Biochemistry:Transport; Nucleic Acids p. 9 of 50 Receptor-mediated endocytosis Bind macromolecule to specific receptor in plasma membrane Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside) Vesicle fuses with endosome and a lysozome Inside the lysozyome, the foreign material and the receptor get degraded … or ligand or receptor or both get recycled 09/30/08 Biochemistry:Transport; Nucleic Acids p. 10 of 50 Example: LDL-cholesterol Diagram courtesy Gwen Childs, U.Arkansas for Medical Sciences 09/30/08 Biochemistry:Transport; Nucleic Acids p. 11 of 50 Exocytosis Materials to be secreted are enclosed in vesicles by the Golgi apparatus Vesicles fuse with plasma membrane Contents released into extracellular space Diagram courtesy LinkPublishing.com 09/30/08 Biochemistry:Transport; Nucleic Acids p. 12 of 50 Transducing signals Plasma membranes contain receptors that allow the cell to respond to chemical stimuli that can’t cross the membrane Bacteria can detect chemicals: if something useful comes along, a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source 09/30/08 Biochemistry:Transport; Nucleic Acids p. 13 of 50 Multicellular signaling Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals 09/30/08 Biochemistry:Transport; Nucleic Acids Diagram courtesy Science Creative Quarterly, U. British Columbia p. 14 of 50 Extracellular Signals Internal behavior of cells modulated by external influences Extracellular signals are called first messengers 7-helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors 09/30/08 Biochemistry:Transport; Nucleic Acids Image courtesy CSU Channel Islands p. 15 of 50 Internal results of signals Intracellular: heterotrimeric G-proteins are the transducers: they receive signal from receptor, hydrolyze GTP, and emit small molecules called second messengers Second messengers diffuse to target organelle or portion of cytoplasm Many signals, many receptors, relatively few second messengers Often there is amplification involved 09/30/08 Biochemistry:Transport; Nucleic Acids p. 16 of 50 Roles of these systems Response to sensory stimuli Response to hormones Response to growth factors Response to some neurotransmitters Metabolite transport Immune response This stuff gets complicated, because the kinds of signals are so varied! 09/30/08 Biochemistry:Transport; Nucleic Acids p. 17 of 50 G proteins Transducers of external signals into the inside of the cell These are GTPases (GTP GDP + Pi) GTP-bound protein transduces signals GDP-bound protein doesn’t Heterotrimeric proteins; association of b and g subunits with a subunit is disrupted by complexation with hormone-receptor complex, allowing departure of GDP & binding of GTP 09/30/08 Biochemistry:Transport; Nucleic Acids p. 18 of 50 GTP G protein cycle Inactive GDP a b Ternary complex g disrupted by binding of b receptor complex g Ga-GTP interacts with effector enzyme GTP slowly hydrolyzed away Then Ga-GDP reassociates with b,g See fig. 9.39 for details 09/30/08 Biochemistry:Transport; Nucleic Acids Active a GTP H2O Pi a GDP Inactive p. 19 of 50 Adenylyl cyclase Cyclic cAMP and cGMP: AMP second messengers Adenylyl cyclase converts ATP to cAMP Integral membrane enzyme; active site faces cytosol cAMP diffuses from membrane surface through cytosol, activates protein kinase A PKA phosphorylates ser,thr in target enzymes; action is reversed by specific phosphatases 09/30/08 Biochemistry:Transport; Nucleic Acids p. 20 of 50 O N N Modulators of cAMP O N N caffeine O N Caffeine, theophylline inhibit cAMP O N phosphodiesterase, prolonging cAMP’s theophylline stimulatory effects on protein kinase A Hormones that bind to stimulatory receptors activate adenylyl cyclase, raising cAMP levels Hormones that bind to inhibitory receptors inhibit adenylyl cyclase activity via receptor interaction with the transducer Gi. 09/30/08 Biochemistry:Transport; Nucleic Acids p. 21 of 50 H N N O O Inositol-Phospholipid Signaling Pathway R1 R2 O O O 2 Second messengers derived from O phosphatidylinositol 4,5-bisphosphate (PIP2) Ligand binds to specific receptor; signal transduced through G protein called Gq Active form activates phosphoinositide-specific phospholipase C bound to cytoplasmic face of plasma membrane 09/30/08 Biochemistry:Transport; Nucleic Acids P O- OH O OH HO O HO -O P O p. 22 of 50 O O -O PIP2 chemistry Phospholipase C hydrolyzes PIP2 to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol Both of these products are second messengers that transmit the signal into the cell OP O P O- O O- O OH -O OP HO O O OH IP3 O O R1 R2 O O OH diacylglycerol 09/30/08 Biochemistry:Transport; Nucleic Acids p. 23 of 50 O -O OP O IP3 and calcium P O- O O OOH -O OP HO O O OH IP3 diffuses through cytosol and binds to a IP3 calcium channel in the membrane of the endoplasmic reticulum The calcium channel opens, releasing Ca2+ from lumen of ER into cytosol Ca2+ is a short-lived 2nd messenger too: it activates Ca2+-dependent protein kinases that catalyze phosphorylation of certain proteins 09/30/08 Biochemistry:Transport; Nucleic Acids p. 24 of 50 O O R1 R2 O O Diacylglycerol and protein kinase C OH diacylglycerol Diacylglycerol stays @ plasma membrane Protein kinase C (which exists in equilibrium between soluble & peripheralmembrane form) moves to inner face of membrane; it binds transiently and is activated by diacylglycerol and Ca2+ Protein kinase C catalyzes phosphorylation of a bunch of proteins 09/30/08 Biochemistry:Transport; Nucleic Acids p. 25 of 50 Control of inositolphospholipid pathway After GTP hydrolysis, Gq is inactive so I no longer stimulates Phospholipase C Activities of 2nd messengers are transient IP3 rapidly hydrolyzed to other things Diacylglycerol is phosphorylated to form phosphatidate 09/30/08 Biochemistry:Transport; Nucleic Acids p. 26 of 50 Sphingolipids give rise to 2nd messengers Some signals activate hydrolases that convert sphingomyelin to sphingosine, sphingosine-1-P, and ceramide Sphingosine inhibits PKC Ceramides activates a protein kinase and a protein phosphatase Sphingosine-1-P can activate Phospholipase D, which catalyzes hydrolysis of phosphatidylcholine; products are 2nd messengers 09/30/08 Biochemistry:Transport; Nucleic Acids p. 27 of 50 ligands exterior Receptor tyrosine kinases Tyr kinase monomers interior Most growth factors function via a pathway that involves these enzymes In absence of ligand, 2 nearby tyr kinase molecules are separated Upon substrate binding they come together, form a dimer 09/30/08 Biochemistry:Transport; Nucleic Acids p. 28 of 50 Autophosphorylation of the dimer P P Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation Once it’s phosphorylated, it’s activate and can phosphorylate various cytosolic proteins, starting a cascade of events 09/30/08 Biochemistry:Transport; Nucleic Acids p. 29 of 50 Insulin receptor Insulin binds to an a2b2 tetramer; binding brings b subunits together Each tyr kinase (b) subunit phosphorylates the other one The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation 09/30/08 Biochemistry:Transport; Nucleic Acids Sketch courtesy of Davidson College, NC p. 30 of 50 6 5 Pyrimidines N 4 1 2 N 3 Single-ring nucleic acid bases pyrimidine 6-atom ring; always two nitrogens in the ring, meta to one another Based on pyrimidine, although pyrimidine itself is not a biologically important molecule Variations depend on oxygens and nitrogens attached to ring carbons Tautomerization possible Note line of symmetry in pyrimidine structure 09/30/08 Biochemistry:Transport; Nucleic Acids p. 31 of 50 H N O Uracil and thymine Uracil is a simple dioxo derivative of pyrimidine: 2,4-dioxopyrimidine Thymine is 5-methyluracil Uracil is found in RNA; Thymine is found in DNA We can draw other tautomers where we move the protons to the oxygens O HN uracil HN O 09/30/08 Biochemistry:Transport; Nucleic Acids N H thymine p. 32 of 50 O Tautomers Lactam and Lactim forms Getting these right was essential to Watson & Crick’s development of the DNA double helical model H N O NH O HN O uracil - lactam H N O uracil - lactim HN HN O N H thymine - lactam O O 09/30/08 Biochemistry:Transport; Nucleic Acids N thymine - lactim p. 33 of 50 OH H N O Cytosine NH2 N cytosine This is 2-oxo,4-aminopyrimidine It’s the other pyrimidine base found in DNA & RNA Spontaneous deamination (CU) we’ll see the significance of that later Again, other tautomers can be drawn 09/30/08 Biochemistry:Transport; Nucleic Acids p. 34 of 50 Cytosine: amino and imino forms Again, this tautomerization needs to be kept in mind H N O NH2 N cytosine -amino form H N O NH N cytosine -imino form 09/30/08 Biochemistry:Transport; Nucleic Acids p. 35 of 50 7 6 5 1N Purines 8 4 2 N N 3 H N 9 Derivatives of purine; again, the purine root molecule isn’t biologically important Six-membered ring looks a lot like pyrimidine Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines 09/30/08 Biochemistry:Transport; Nucleic Acids p. 36 of 50 Adenine This is 6-aminopurine Found in RNA and DNA We’ve seen how important adenosine and its derivatives are in metabolism Tautomerization happens here too NH NH2 H N N N N adenine - amino form H N HN N N adenine - imino form 09/30/08 Biochemistry:Transport; Nucleic Acids p. 37 of 50 Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms OH O H N H N N HN H2N N guanine - lactam N H2N N N guanine - lactim p. 38 of 50 09/30/08 Biochemistry:Transport; Nucleic Acids Other natural purines Hypoxanthine and xanthine are biosynthetic precursors of A & G Urate is important in nitrogen excretion pathways 09/30/08 Biochemistry:Transport; Nucleic Acids p. 39 of 50 Tautomerization and H-bonds Lactam forms predominate at neutral pH This influences which bases are H-bond donors or acceptors Amino groups in C, A, G make H-bonds So do ring nitrogens at 3 in pyrimidines and 1 in purines … and oxygens at 4 in U,T, 2 in C, 6 in G 09/30/08 Biochemistry:Transport; Nucleic Acids p. 40 of 50 O HO Nucleosides NR1R2 OH HO N-glycoside of ribofuranose As mentioned in ch. 8, these are glycosides of the nucleic acid bases Sugar is always ribose or deoxyribose Connected nitrogen is: N1 for pyrimidines (on 6-membered ring) N9 for purines (on 5-membered ring) 09/30/08 Biochemistry:Transport; Nucleic Acids p. 41 of 50 Pyrimidine nucleosides Drawn here in amino and lactam forms OH OH HO HO OH O N H2N N OH O cytidine O N O N H O uridine 09/30/08 Biochemistry:Transport; Nucleic Acids p. 42 of 50 OH Pyrimidine deoxynucleosides OH H H OH O N O N H OH O OH 2'-deoxyuridine O N H OH N O 2'-deoxythymidine O N H2N O N O deoxycytidine 09/30/08 Biochemistry:Transport; Nucleic Acids p. 43 of 50 A tricky nomenclature issue Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed. 09/30/08 Biochemistry:Transport; Nucleic Acids p. 44 of 50 Purine nucleosides Drawn in amino and lactam forms NH2 O N N N HN N N H2N N N O O HO HO OH OH HO adenosine HO guanosine 09/30/08 Biochemistry:Transport; Nucleic Acids p. 45 of 50 Purine deoxynucleosides O NH2 N N HN N N H2N N N N O O OH OH HO HO deoxyadenosine deoxyguanosine 09/30/08 Biochemistry:Transport; Nucleic Acids p. 46 of 50 Conformations around the glycosidic bond Rotation of the base around the glycosidic bond is sterically hindered In the syn conformation there would be some interference between the base and the 2’hydroxyl of the sugar Therefore pyrimidines are always anti, and purines are usually anti Furanose and base rings are roughly perpendicular 09/30/08 Biochemistry:Transport; Nucleic Acids p. 47 of 50 Glycosidic bonds This illustrates the roughly perpendicular positionings of the base and sugar rings 09/30/08 Biochemistry:Transport; Nucleic Acids p. 48 of 50 Solubility of nucleosides and lability of glycosidic linkages The sugar makes nucleosides more soluble than the free bases Nucleosides are generally stable to basic hydrolysis Acid hydrolysis: Purines: glycosidic bond fairly readily hydrolized Pyrimidines: resistant to acid hydrolysis 09/30/08 Biochemistry:Transport; Nucleic Acids p. 49 of 50 Chirality in nucleic acids Bases themselves are achiral Four asymmetric centers in ribofuranose, counting the glycosidic bond. Three in deoxyribofuranose Glycosidic bond is one of those 4 or 3. Same for nucleotides: phosphates don’t add asymmetries 09/30/08 Biochemistry:Transport; Nucleic Acids p. 50 of 50