Transport & Signaling; Nucleic Acid Chemistry

1. Transport & Signaling;Nucleic Acid Chemistry Andy HowardIntroductory Biochemistry30 September 2008 Biochemistry:Transport; Nucleic Acids

2. Membrane transport Energetics Active transport Transporting big molecules Membrane Signaling Adenylyl cyclase Inositol-phospholipid signaling pathway Receptor tyr kinases Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G Nucleosides What we’ll discuss Biochemistry:Transport; Nucleic Acids

3. 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 Diagram courtesy Saint-Boniface U. Biochemistry:Transport; Nucleic Acids

4. 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. Biochemistry:Transport; Nucleic Acids

5. Primary active transport • Energy source:usually ATP or light • Energy source directlycontributes to overcomingconcentration gradient • 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 BacteriorhodopsinPDB 1F50, 1.7Å25 kDa monomer Biochemistry:Transport; Nucleic Acids

6. Secondary active transport • Active transport of one solute is coupled to passive transport of another • Net energetics is (just barely) favorable • Generally involves antiport • Bacterial lactose influx driven by proton efflux • Sodium gradient often used in animals Pyrococcus Multi-sugar transporterPDB 1VCI83 kDa dimer Biochemistry:Transport; Nucleic Acids

7. Complex case: Na+/K+ pump • Typically [Kin] = 140mM, [Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM. • ATP-driven transporter:3 Na+ out for 2 K+ inper 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 Diagram courtesy Steve Cook Biochemistry:Transport; Nucleic Acids

8. 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 Diagram courtesy Steve Cook Biochemistry:Transport; Nucleic Acids

9. 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 Biochemistry:Transport; Nucleic Acids

10. 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 Biochemistry:Transport; Nucleic Acids

11. Example: LDL-cholesterol Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences Biochemistry:Transport; Nucleic Acids

12. Exocytosis Diagram courtesy LinkPublishing.com • Materials to be secreted are enclosed in vesicles by the Golgi apparatus • Vesicles fuse with plasma membrane • Contents released into extracellular space Biochemistry:Transport; Nucleic Acids

13. 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 Biochemistry:Transport; Nucleic Acids

14. Multicellular signaling • Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals Diagram courtesy Science Creative Quarterly, U. British Columbia Biochemistry:Transport; Nucleic Acids

15. Extracellular Signals • Internal behavior ofcells 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 Image courtesy CSU Channel Islands Biochemistry:Transport; Nucleic Acids

16. 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 Biochemistry:Transport; Nucleic Acids

17. 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! Biochemistry:Transport; Nucleic Acids

18. G proteins • Transducers of external signals into the inside of the cell • These are GTPases (GTP  GDP + Pi) • GTP-bound protein transduces signalsGDP-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 Biochemistry:Transport; Nucleic Acids

19. GTP Inactive GDP G protein cycle a Active b a g GTP b • Ternary complex disrupted by binding of receptor complex • Ga-GTP interacts with effector enzyme • GTP slowly hydrolyzed away • Then Ga-GDP reassociates with b,g • See fig. 9.39 for details H2O g Pi a GDP Inactive Biochemistry:Transport; Nucleic Acids

20. Adenylyl cyclase Cyclic AMP • cAMP and cGMP: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 Biochemistry:Transport; Nucleic Acids

21. Modulators of cAMP • Caffeine, theophylline inhibit cAMP phosphodiesterase, prolonging cAMP’s 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. Biochemistry:Transport; Nucleic Acids

22. Inositol-Phospholipid Signaling Pathway • 2 Second messengers derived from 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 Biochemistry:Transport; Nucleic Acids

23. 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 Biochemistry:Transport; Nucleic Acids

24. IP3 and calcium • IP3 diffuses through cytosol and binds to a 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 Biochemistry:Transport; Nucleic Acids

25. Diacylglycerol and protein kinase C • Diacylglycerol stays @ plasma membrane • Protein kinase C (which exists in equilibrium between soluble & peripheral-membrane 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 Biochemistry:Transport; Nucleic Acids

26. Control of inositol-phospholipid 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 Biochemistry:Transport; Nucleic Acids

27. 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 Biochemistry:Transport; Nucleic Acids

28. 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 Biochemistry:Transport; Nucleic Acids

29. 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 Biochemistry:Transport; Nucleic Acids

30. 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 Sketch courtesy ofDavidson College, NC Biochemistry:Transport; Nucleic Acids

31. 6 1 5 Pyrimidines 4 2 3 • Single-ring nucleic acid bases • 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 Biochemistry:Transport; Nucleic Acids

32. 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 Biochemistry:Transport; Nucleic Acids

33. Tautomers • Lactam and Lactim forms • Getting these right was essential to Watson & Crick’s development of the DNA double helical model Biochemistry:Transport; Nucleic Acids

34. Cytosine • This is 2-oxo,4-aminopyrimidine • It’s the other pyrimidine base found in DNA & RNA • Spontaneous deamination (CU)we’ll see the significance of that later • Again, other tautomers can be drawn Biochemistry:Transport; Nucleic Acids

35. Cytosine:amino and imino forms • Again, this tautomerization needs to be kept in mind Biochemistry:Transport; Nucleic Acids

36. 7 6 5 1 8 4 Purines 2 9 3 • Derivatives of purine; again, the 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 Biochemistry:Transport; Nucleic Acids

37. 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 Biochemistry:Transport; Nucleic Acids

38. Guanine • This is 2-amino-6-oxopurine • Found in RNA, DNA • Lactam, lactim forms Biochemistry:Transport; Nucleic Acids

39. Other natural purines • Hypoxanthine and xanthine are biosynthetic precursors of A & G • Urate is important in nitrogen excretion pathways Biochemistry:Transport; Nucleic Acids

40. 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 Biochemistry:Transport; Nucleic Acids

41. Nucleosides • 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) Biochemistry:Transport; Nucleic Acids

42. Pyrimidine nucleosides • Drawn here in amino and lactam forms Biochemistry:Transport; Nucleic Acids

43. Pyrimidine deoxynucleosides Biochemistry:Transport; Nucleic Acids

44. 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. Biochemistry:Transport; Nucleic Acids

45. Purine nucleosides • Drawn in amino and lactam forms Biochemistry:Transport; Nucleic Acids

46. Purine deoxynucleosides Biochemistry:Transport; Nucleic Acids

47. 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 Biochemistry:Transport; Nucleic Acids

48. Glycosidic bonds • This illustrates the roughly perpendicular positionings of the base and sugar rings Biochemistry:Transport; Nucleic Acids

49. 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 Biochemistry:Transport; Nucleic Acids

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 Biochemistry:Transport; Nucleic Acids