1. Enzymes and transporters a review

2. Protein-Ligand Interactions

3. Learning Objectives Know the importance and general characteristics of protein-ligand interactions, including: – Enzyme-substrates – Substrates-transporters/channels – Hormones-receptors – Complex signal transduction mechanisms Be able to interpret the factors that influence the rates of reaction of enzyme (or more generally the rate at which processes mediated by protein-ligand interactions take place). Know the mechanisms by which different materials (water and solutes are transported across biological membranes) including the differences among: – Simple diffusion, channels, facilitated transport, active transport Know the mechanisms by which transport across biological membranes is regulated. Know the mechanisms by which cells communicate with other cells including: – Direct communication across gap junctions, autocrine, paracrine, and endocrine Know the mechanisms by which membrane and cytoplasmic receptors function including: G-proteins and enzyme-linked receptors.

4. examples of protein-ligand interactions? Enzymes Channels Transporters Pumps (active transporters) Receptors Others Chaperones Gas carriers Antibodies

5. Enzymes

8. Dehydration synthesis and hydrolysis Catalyzed by a “hydrolase” Catalyzed by a “synthase” Note the “ase”

9. HOMEWORK Please find examples of dehydration synthesis and hydrolysis (say 5 of each).

10. To Remember Protein ligand interactions mediate most of the processes that take place in cells (indeed in living systems) Protein ligand interaction involve the formation of a protein ligand complex and its dissociation into the protein and a product (or process). Proteins involved in protein ligand interactions often have one (or more) binding sites. Important examples of protein-ligand interactions are enzymes, transporters, receptors, chaperone proteins, and transport proteins.

11. About enzymes Enzymes are proteins Enzymes are biological catalysts Enzymes participate in both “catalysis” (breakdown) and “synthesis” Enzymes have active sites

12. About enzymes Enzymes are proteins Enzymes are biological catalysts Enzymes participate in both “catalysis” (breakdown) and “synthesis” Enzymes have active sites The activity of enzymes depends on temperature, pH, and ….

15. In humans pepsin acts on the nutrients found in the contents of the _____________, whereas trypsin acts in the nutrients found in the contents of the ________________ a)Both enzymes act within the stomach b)Both enzymes act within the small intestine c)stomach, small intestine d)intestine, stomach The duodenum, jejunium, and ileum, are sections of the small intestine.

16. About enzymes Enzymes are proteins Enzymes are biological catalysts Enzymes participate in both “catalysis” (breakdown) and “synthesis” Enzymes have active sites The activity of enzymes depends on temperature, pH, and substrate concentration

18. Enzyme Kinetics

20. Message (TO REMEMBER) The rate at which processes mediated by protein-ligand interactions takes place saturates.

21. About enzymes Enzymes are proteins Enzymes are biological catalysts Enzymes participate in both “catalysis” (breakdown) and “synthesis” Enzymes have active sites The activity of enzymes depends on temperature, pH, and substrate concentration The activity of enzymes in a cell can be modulated (varied)

24. A very important mechanism in the regulation/modulation of enzyme activity is by phosphorylation. Enzymes that activate an enzyme by adding a phosphate group are called phosphotransferaces or kinases, those that deactivate it by removing a phosphate group are called phosphatases.

26. A reminder …..

27. TO REMEMBER Enzymes are proteins Enzymes are biological catalysts Enzymes participate in both “catalysis” (breakdown) and “synthesis” Enzymes have active sites The activity of enzymes depends on temperature, pH, and substrate concentration The activity of enzymes in a cell can be modulated (varied) by allosteric modulation (phosphorylation is an example)

28. PFK (phosphofructokinase) catalyzes the third step of glycolysis. If the levels of ATP in the cell are very high, ATP will bind to a site in PFK and inhibit it. The result is reduction in the rate of glycolysis. This is an example of a) dehydration synthesis b) hydrolysis c) allosteric inhibition d) competitive facilitation

29. Transport Across Cell Membranes

30. About Transport Across Membrane Solutes can move across membranes by Simple Diffusion (only lipophylic/non- polar substrates such as ethanol)

31. Simple Diffusion always takes place down-hill an electrochemical gradient and is non-saturable and non- concentrative.

32. About Transport Across Membranes Solutes can move across membranes by Simple Diffusion (only lipophylic/non- polar substrates) Across Channels (like aquaporins)

34. BIG MESSAGE WATER IS TRANSPORTED ACROSS CELL MEMBRANES THROUGH AQUAPORINS (Many types)

35. About Transport Across Membranes I Solutes can move across membranes by Simple Diffusion (only lipophylic/non- polar substrates, ETOH) Across Channels (like aquaporins, water) Channels can be “gated” (that is they can open and close as a result of stimuli)

36. About Transport Across Membranes I Solutes can move across membranes by Simple Diffusion (only lipophylic/non- polar substrates, ETOH) Across Channels (like aquaporins, water) Channels can be “gated” (that is they can open and close as a result of stimuli)

37. The opening and closing of channels can be a form of allosteric modulation

39. Channels can be gated by changes in Membrane potential (voltage gated) Ligands (ligand gated) Mechanical stimulation (stress gated)

40. About Transport Across Membranes Solutes can move across membranes by Simple Diffusion (only lipophylic/non-polar substrates) Across Channels (like aquaporins) Channels can be “gated” (that is they can open and close as a result of stimuli) The stimuli that open and close channels can be: electrical charge across the membrane (voltage gated), binding with a ligand (ligand gated), and mechanical stimulation (stress activated)

41. Transport Across Cell Membranes

42. About Transport Across Membrane Solutes can move across membranes by Carrier Mediated Diffusion Carrier Mediated Diffusion is NOT concentrative and is “downhill” (it take place only when there is a solute concentration gradient across the membrane). It DOES NOT require energy

45. The transport of glucose into red blood cells (erythrocytes) can only take place when the concentration of glucose in plasma is higher than that inside of the erythrocyte. Transport rate saturates, and does not require energy. This is an example of a)passive diffusion b) a voltage gated channel c)an aquaporin d)Facilitated transport

46. About Transport Across Membranes Solutes can move across membranes by Carrier Mediated Diffusion Active transport Active Transport -IS concentrative and can be “uphill” (it can take place against concentration gradient across the membrane). -It requires energy -It is “saturable”

47. The sodium-potasium ATP-ase pump

49. The Sodium-Potassium Pump (Na+/K+ - ATPase) - A case of PRIMARY active transport This is one of the best examples of active transport in animal cells -This pump transports Na+ ions out of the cell and K+ ions into the cell. Thus keeping the intracellular concentration of Na low compared to outside, and the intracellular concentration of K high -The pump is driven by hydrolysis of ATP -It uses about 30% of the energy available to any one animal cell! -The pump is a transmembrane carrier protein made up of 4 subunits (2 large and 2 small)

50. The Sodium-Potassium Pump (Na+/K+ - ATPase) I - A case of PRIMARY active transport This is one of the best examples of active transport in animal cells -This pump transports Na+ ions out of the cell and K+ ions into the cell. Thus keeping the intracellular concentration of Na low compared to outside, and the intracellular concentration of K high -The pump is driven by hydrolysis of ATP -It uses about 30% of the energy available to any one animal cell! -The pump is a transmembrane carrier protein made up of 4 subunits (2 large and 2 small)

51. The Sodium-Potassium Pump (Na+/K+ - ATPase) II -Structure: It has 3 binding sites for sodium ions, 2 for potasium ions, and one phosphorylation site to accept a phosphate from ATP. -Hydrolysis of one ATP molecule fuels the export of 3 Na+ ions out of the cell and 2 K+ ions into the cell.

52. Primary Active Transport In PRIMARY ACTIVE transport, the site of transport and the ATPase are together. The transport of the solute and the hydrolysis of ATP are coupled. WE WILL TALK ABOUT SECONDARY ACTIVE TRANSPORT LATER

53. About Transport Across Membranes (TO REMEMBER) Solutes can move across membranes by Simple Diffusion (only lipophylic/non-polar substrates) Across Channels (like aquaporins) Channels can be “gated” (that is they can open and close as a result of stimuli) The stimuli that open and close channels can be: electrical charge across the membrane (voltage gated), binding with a ligand (ligand gated), and mechanical stimulation (stress activated)

54. About Transport Across Membrane (TO REMEMBER) Solutes can move across membranes by Carrier Mediated Diffusion Active Transport – Primary Active (Sodium-Potasium ATPase is an example) – Secondary Active (LATER)

55. TO REMEMBER Kinds of Transport No Help Simple Diffusion O 2, CO 2, and ETOH (non-polar) Membrane Proteins Channels Aquaporins (H 2 O) Ion Channels (Na+, K+, Cl-, Ca++) Carriers/ Transporters Glucose, amino acids (large, polar) Pumps Na+/K+ ATPase H+ (Proton) Vesicles Endocytosis Exocytosis SATURABLE ACTIVE

56. Endocytocis and Exocytosis

57. Both endocytosis and exocytosis require energy because vesicles must be moved into and out of the membrane using microtubules as scaffold. They are examples of active transport!!!

58. TO REMEMBER Kinds of Transport No Help Simple Diffusion O 2, CO 2, and ETOH (non-polar) Membrane Proteins Channels Aquaporins (H 2 O) Ion Channels (Na+, K+, Cl-, Ca++) Carriers/ Transporters Glucose, amino acids (large, polar) Pumps Na+/K+ ATPase H+ (Proton) Vesicles Endocytosis Exocytosis SATURABLE ACTIVE

60. Receptors and Cell Communication (FIVE ways of sending a message)

61. ONE MORE (AN IMPORTANT ONE)

62. REMINDER

63. CONNEXINS

64. Examples (across gap junctions) -allow direct communication among cells -by transmission of small molecules (calcium and secondary messengers)

65. Examples (paracrine signaling)

67. Examples (endocrine signaling) All hormones

68. In Summary and TO REMEMBER Cells send signals to other cells By direct exchange of signaling molecules (calcium and secondary messengers) across gap junctions By secreting a signaling molecule into adjacent cells (paracrine signaling including synaptic signaling) By secreting a signaling molecule into the circulatory system so that it reaches the target cell (endocrine signaling)

69. Cells receive chemical signals from other cells by binding these signals to specific receptors.

70. Receptors come in many forms, but two general types 1) If the signaling molecule cannot cross the membrane (because it is polar and lipid insoluble), then the receptor is in the membrane. In this case, the response is rapid, mediated by signal amplification (activation of enzymes), and often (but not always) transient (i.e. short lived).

71. Receptors come in many forms, but two general types 2) If the signaling molecule crosses the membrane (because it is lipid soluble), then the receptor is inside of the cell the membrane. In this case, the response is slower, mediated by protein synthesis, and often (but not always) persistent (i.e. long lived).

72. In Summary and TO REMEMBER Cells RECEIVE chemical signals from other cells By binding these signaling molecules to receptors that can be In the membrane -if the signaling molecule is not lipid soluble -in this case, the response is fast, mediated by a metabolic activation cascade, and often transient Within the cell – If the signaling molecule crosses the membrane -in this case the response is slower, mediated by protein synthesis, and often persistent

73. STEROID HORMONES (SECRETED BY THE ADRENAL GLAND) HAVE INTRACELLULAR RECEPTORS

75. Many (BUT NOT ALL) membrane receptors act through G- proteins

76. SECONDARY MESSENGER

77. SIGNAL AMPLIFICATION 1 1000 10,000 1,000,000

78. NOT ALL MEMBRANE RECEPTORS ACT THROUGH G-PROTEINS

79. In Summary and TO REMEMBER Membrane receptors can be either enzyme-linked or act by G-proteins. In either case they often act through a phosphorylation amplification cascade. Because G-proteins activate adenyl- cyclase, in the case of G-protein receptors, the secondary messenger is cyclic AMP.

80. REVIEW QUESTIONS 1)Describe all the kinds of protein ligand interactions that you can (at least 5). 2)What does it mean that a protein-ligand interaction is “saturable”. Give at least 3 examples of saturation in protein ligand interactions. 3)Describe the most common form of allosteric modulation (phosphorylation and dephosphorylation) 4)Why do we call kinases “kinases”? 5)Construct a table describing ALL the mechanisms by which molecules are transported across biological membranes and their properties (type of molecule transported, saturable or not, and whether they use energy or not)

81. REVIEW QUESTIONS 6) What are the stimuli for the opening/closing of voltage gated, ligand gated, and stress gated channels. 8) What are the differences in how molecules are transported by carrier-mediated facilitated diffusion and simple diffusion. 7) Describe in detail the mechanism by which the Na+/K+ ATPase pump moves Na+ and K+ out and into of cells. 8) What do the terms autocrine, paracrine, and endocrine mean? 9) Why is it that if a messenger/hormone has intracellular receptors, its effect are long-lasting/persistent.

82. REVIEW QUESTIONS 10) Why is it that when the receptor is at the cell membrane, the effect on the target cell is usually rapid and persistent. 11) Construct (without looking at notes) a diagram showing how G- proteins work.