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Protein Functions II; Intro to Carbohydrates

Protein Functions II; Intro to Carbohydrates. Andy Howard Introductory Biochemistry, Fall 2009 17 September 2009. Proteins and carbohydrates. Proteins perform a variety of functions, including acting as enzymes.

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Protein Functions II; Intro to Carbohydrates

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  1. Protein Functions II;Intro to Carbohydrates Andy HowardIntroductory Biochemistry, Fall 2009 17 September 2009 Biochem: Functions II; Carbohydrates I

  2. Proteins and carbohydrates • Proteins perform a variety of functions, including acting as enzymes. • Sugars are vital as energy sources, and they also serve as building blocks for lipid-carbohydrate and protein-carbohydrate complexes Biochem: Functions II; Carbohydrates I

  3. Classes of proteins and their roles Enzyme properties Classes of enzymes Classification schemes Sugar Concepts Review of chirality Monosaccharides Oligosaccharides Glycosides Plans for Today Biochem: Functions II; Carbohydrates I

  4. Classes of proteins Arginosuccinate lyase / Delta crystallinPDB 1auw, 2.5Å206kDa tetramer • Reminder:proteins can take onmore than one function • A protein may evolve for one purpose • … then it gets co-opted for another • Moonlighting proteins (Jeffery et al, Tobeck) Biochem: Functions II; Carbohydrates I

  5. Structural proteins • Perform mechanical or scaffolding tasks • Not involved in chemistry, unless you consider this to be a chemical reaction:(Person standing upright) (Person lying in a puddle on the floor) • Examples: collagen, fibroin, keratin • Often enzymes are recruited to perform structural roles CollagenmodelPDB 1K6F Biochem: Functions II; Carbohydrates I

  6. Enzymes • Enzymes are biological catalysts, i.e. their job is to reduce the activation energy barrier between substrates and products • Tend to be at least 12kDa (why?You need that much scaffolding) • Usually but not always aqueous • Usually organized with hydrophilic residues facing outward hen egg-white lysozyme PDB 2vb10.65Å, 14.2kDa Biochem: Functions II; Carbohydrates I

  7. Many enzymes are oligomeric PDB 2hcy: tetramer • Both heterooligomers and homooligomers • ADH: tetramer of identical subunits • RuBisCO: 8 identical large subunits, 8 identical small subunits PDB 1ej7: 2.45Å8*(13.5+52.2kDa) Biochem: Functions II; Carbohydrates I

  8. IUBMB Major Enzyme Classes Biochem: Functions II; Carbohydrates I

  9. Enzymes have 3 features • Catalytic power (they lower G‡) • Specificity • They prefer one substrate over others • Side reactions are minimized • Regulation • Can be sped up or slowed down by inhibitors and accelerators • Other control mechanisms exist Biochem: Functions II; Carbohydrates I

  10. EC System Porcine pancreatic elastasePDB 3EST 1.65 Å 26kDa monomer • 4-component naming system,sort of like an internet address • Pancreatic elastase: • Category 3: hydrolases • Subcategory 3.4: hydrolases acting on peptide bonds (peptidases) • Sub-subcategory 3.4.21: Serine endopeptidases • Sub-sub-subcategory 3.4.21.36: Pancreatic elastase Biochem: Functions II; Carbohydrates I

  11. Category 1:Oxidoreductases • General reaction:Aox + Bred Ared + Box • One reactant often a cofactor (see ch.7) • Cofactors may be organic (NAD or FAD)or metal ions complexed to proteins • Typical reaction:H-X-OH + NAD+ X=O + NADH + H+ Biochem: Functions II; Carbohydrates I

  12. Category 2:Transferases • These catalyze transfers ofgroups like phosphate or amines. • Example: L-alanine + a-ketoglutarate pyruvate + L-glutamate • Kinases are transferases:they transfer a phosphate from ATP to something else Biochem: Functions II; Carbohydrates I

  13. O O- Category 3:hydrolases HO-P-O-P-OH O O- • Water is acceptor of transferred group • Ultrasimple: pyrophosphatase:Pyrophosphate + H2O  2 Phosphate • Proteases, lipases, many other sub-categories Biochem: Functions II; Carbohydrates I

  14. C=C Category 4:Lyases • Non-hydrolytic, nonoxidative elimination (or addition) reactions • Addition across a double bond or reverse • Example: pyruvate decarboxylase:pyruvate + H+ acetaldehyde + CO2 • More typical lyases add across C=C Biochem: Functions II; Carbohydrates I

  15. Category 5: Isomerases • Unimolecular interconversions(glucose-6-P  fructose-6-P) • Reactions usually almost exactly isoergic • Subcategories: • Racemases: alter stereospecificity such that the product is the enantiomer of the substrate • Mutases: shift a single functional group from one carbon to another (phosphoglucomutase) Biochem: Functions II; Carbohydrates I

  16. Category 6: Ligases • Catalyze joining of 2 substrates,e.g.L-glutamate + ATP + NH4+L-glutamine + ADP + Pi • Require input of energy from XTP (X=A,G) • Usually called synthetases(not synthases, which are lyases, category 4) • Typically the hydrolyzed phosphate is not incorporated into the product Biochem: Functions II; Carbohydrates I

  17. iClicker quiz, question 1 • Collagen is a structural protein. Collagenase catalyzes the hydrolysis of collagen under appropriate circumstances. It is: • (a) an enzyme • (b) a hormone • (c) a receptor • (d) a nucleic-acid binding protein • (e) there’s no way to tell from the information provided. Biochem: Functions II; Carbohydrates I

  18. iClicker quiz, question 2 • Suppose a membrane protein has 4 transmembrane helices and 2 aqueous domains, one at the N-terminal end and the other at the C-terminal end. Assuming the N-terminal aqueous domain is in the cytoplasm, where would the C-terminal aqueous domain be?(a) in the cytoplasm(b) in the membrane(c) in the extracellular space(d) no way to tell Biochem: Functions II; Carbohydrates I

  19. iClicker quiz, question 3 • Which IUBMB enzyme category would collagenase fall into? • (a) ligases (6) • (b) oxidoreductases (1) • (c) hydrolases (3) • (d) isomerases (5) • (e) none of the above. Biochem: Functions II; Carbohydrates I

  20. iClicker quiz, question 4 • Triosephosphate isomerase, whose structure we discussed last week, interconverts glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. What would you expect the approximate DG value for this reaction to be? • (a) -30 kJ mol-1 (d) 0 kJ mol-1 • (b) 30 kJ mol-1 (e) no way to tell. • (c) -14 kJ mol-1. Biochem: Functions II; Carbohydrates I

  21. Electron-transport proteins • Involved in Oxidation-reductionreactions via • Incorporated metal ions • Small organic moieties (NAD, FAD) • Generally not enzymes because they’re ultimately altered by the reactions in which they participate • But they can be considered to participate in larger enzyme complexes than can restore them to their original state Recombinant human cytochrome cPDB 1J3SNMR structure11.4kDa Biochem: Functions II; Carbohydrates I

  22. Sizes and characteristics • Some ET proteins: fairly small • Cytochrome c • Some flavodoxins • Others are multi-polypeptide complexes • Cofactors or metals may be closely associated (covalent in cytochromes) or more loosely bound Anacystisflavodoxin PDB 1czn1.7Å18.6 kDa Biochem: Functions II; Carbohydrates I

  23. Storage and transport proteins • Hemoglobin, myoglobin classic examples • “honorary enzymes”: share some characteristics with enzymes • Sizes vary widely • Many transporters operate over much smaller size-scales than hemoglobin(µm vs. m): often involved in transport across membranes • We’ll discuss intracellular transport a lot! Sperm-whale myoglobinPDB 1MTJ 1.7Å 18kDa monomer Biochem: Functions II; Carbohydrates I

  24. Why do we have storage proteins? • Many metabolites are toxic in the wrong places or at the wrong times • Oxygen is nasty • Too much Ca2+ or Fe3+ can be hazardous • So storage proteins provide ways of encapsulating small molecules until they’re needed; then they’re released T.maritimaferritinPDB 1z4a8*(18 kDa) Biochem: Functions II; Carbohydrates I

  25. Hormones Human insulinPDB 1t1k 3.3+2.3 kDa • Transported signaling molecules,secreted by one tissue and detectedby receptors in another tissue • Signal noted by the receptor will trigger some kind of response in the second tissue. • They’re involved in cell-cell or tissue-to-tissue communication. Biochem: Functions II; Carbohydrates I

  26. Not all hormones are proteins • Some are organic, non-peptidic moieties • Others: peptide oligomers, too small to be proteins • Oxytocin: CYIQNCPLG • Angiotensin I: DRVYIHPFHL • Some are cyclic (COO- terminus and NH3+ termini hydrolytically ligated) • But some hormones are in fact normal-sized proteins. Biochem: Functions II; Carbohydrates I

  27. Receptors • Many kinds, as distinguished by what they bind: • Some bind hormones, others metabolites, others non-hormonal proteins • Usually membrane-associated: • a soluble piece sticking out • Hydrophobic piece in the membrane • sometimes another piece on the other side of the membrane • Membrane part often helical:usually odd # of spanning helices (7?) Retinal from bacteriorhodopsinPDB 1r2nNMR structure27.4 kDa Biochem: Functions II; Carbohydrates I

  28. Why should it work this way? • Two aqueous domains, one near N terminus and the other near the C terminus, are separated by an odd number of helices • This puts them on opposite sides of the membrane! Biochem: Functions II; Carbohydrates I

  29. Nucleic-acid binding proteins • Many enzymes interact with RNA or DNA • But there are non-catalytic proteins that also bind nucleic acids Human hDim1PDB 1pqnNMR struct.14kDa Biochem: Functions II; Carbohydrates I

  30. Non-catalytic nucleic acid binding proteins • Scaffolding for ribosomal activity • Help form molecular machines for replication, transcription, RNA processing: • These often involve interactions with specific bases, not just general feel-good interactions • Describe these as “recognition steps” Biochem: Functions II; Carbohydrates I

  31. Scaffolding(adapter) proteins Human regulatory complex(Crk SH2 + Abl SH3)PDB 1JU5NMR structure • A type of signaling protein(like hormones and receptors) • Specific modules of the protein recognize and bind other proteins:protein-protein interactions • They thereby function as scaffolds on which a set of other proteins can attach and work together Biochem: Functions II; Carbohydrates I

  32. Protective proteins E5 Fragment of bovine fibrinogenPDB 1JY2, 1.4Å2*(5.3+6.2+5.8) kDa • Eukaryotic protective proteins: • Immunoglobulins • Blood-clotting proteins(activated by proteolytic cleavage) • Antifreeze proteins Biochem: Functions II; Carbohydrates I

  33. Other protective and exploitive proteins Vibrio cholerae toxin A1 + ARF6PDB 2A5F2.1Å21.2+19.3 kDa • Plant, bacterial, and snake-venom toxins • Ricin, abrin (plant proteins that discourage predation by herbivores) Synthetic Abrin-APDB 1ABR2.14Å29.3+27.6 kDa Biochem: Functions II; Carbohydrates I

  34. Special functions Dioscoreophyllum Monellin PDB 1KRL5.5+4.8 kDa • Monellin: sweet protein • Resilin: ultra-elastic insect wing protein • Glue proteins (barnacles, mussels) • Adhesive ability derived from DOPA crosslinks • Potential use in wound closure! Biochem: Functions II; Carbohydrates I

  35. What percentages do what? • 42% of all human proteins have unknown function! • Enzymes are about 20% of proteins with known functions (incl. 3% kinases, 7.5% nucleic acid enzymes) • Structural proteins 4.2% • Percentages here reflect diversity, not mass Biochem: Functions II; Carbohydrates I

  36. Fig.15 from Venter et al. (2001), Science291:1304 Protein Functions Biochem: Functions II; Carbohydrates I

  37. Carbohydrates • These are polyhydroxylated aldehydes and ketones, many of which can exist in cyclic forms • General monomeric formula (CH2O)m, 3 m 9 • With one exception (dihydroxyacetone) they contain chiral centers • Monomers and small oligomers: highly soluble • Can be oligomerized and polymerized • Oligomers may or may not be soluble • Most abundant organic molecules on the planet Biochem: Functions II; Carbohydrates I

  38. How do we measure solubility for very soluble compounds? • (Note: this is not a serious chemical topic: it’s an example of how statistics can be abused…) • The assertion is that, with highly soluble compounds like sugars, it’s difficult to use conventional approaches to compare their solubilities • The suggestion is that we might use the amount of time it takes to dissolve (for example) 50g of solute in 100mL of cold water: if it’s fast, the solute is more soluble than if it’s slow. Biochem: Functions II; Carbohydrates I

  39. Solubility measured by dissolution time 6 • Assertion: more polar groups means shorter dissolution time for a given class of compounds 5 4 3 # of polar groups 2 1 Time required for dissolution Biochem: Functions II; Carbohydrates I

  40. What if we extrapolate to n=6? Extrapolated point 6 • We get a negative dissolution time! • That is, the solid goes into solution 6 seconds before we put it in the water! • This causes serious psychological problems (what if I change my mind?) and philosophical problems (is this pre-ordained?) 5 4 3 # of polar groups Observed points 2 1 Time required for dissolution Biochem: Functions II; Carbohydrates I

  41. Whose idea is this? • Isaac Asimov, that’s who! • “The endochronic properties of resublimated thiotimolene”:Astounding Science Fiction, March1948 • My point: extrapolations and other misuses of statistics are dangerous • Benjamin Disraeli (popularized by Mark Twain):There are three kinds of untruth:lies, damn lies, and statistics. • Okay: let’s get back to the science. Biochem: Functions II; Carbohydrates I

  42. Aldoses & ketoses • If the carbonyl moiety is at the end carbon (conventionally counted as 1), it’s an aldose • If carbonyl is one carbon away (counted as 2), it’s a ketose • If it’s two or more carbons from the end of the chain, it’s not a sugar Biochem: Functions II; Carbohydrates I

  43. Simplest monosaccharides • Glyceraldehyde and dihydroxyacetone • Only glyceraldehyde is chiral:D-enantiomer is more plentiful in biosphere • All longer sugars can be regarded as being built up by adding-(CHOH)m-1 to either glyceraldehyde or dihydroxyacetone, just below C2 Biochem: Functions II; Carbohydrates I

  44. How many aldoses are there? • Every -(CHOH) in the interior offers one chiral center • An m-carbon aldose has (m-2) internal -(CHOH) groups • Therefore: 2m-2 aldoses of length m • For m=3, that’s 21=2; for m=6, it’s 24=16. Biochem: Functions II; Carbohydrates I

  45. How many ketoses are there? • Every -(CHOH) in the interior offers one chiral center • An m-carbon ketose has (m-3) internal-(CHOH) groups • Therefore: 2m-3 ketoses of length m • For m=3, that’s 20 = 1; for m=6, that’s 23=8. Biochem: Functions II; Carbohydrates I

  46. Review: stereochemical nomenclature • Stereoisomers: compounds with identical covalent bonding apart from chiral connectivity • Enantiomers: compounds for which the opposite chirality applies at all chiral centers • Epimers: compounds that differ in chirality at exactly one chiral center • One chiral center: enantiomers are epimers. • > 1 chiral center: enantiomers are not epimers. Biochem: Functions II; Carbohydrates I

  47. Example: 2 chiral centers • Chiral centers u,v; compounds A,B,C,D Biochem: Functions II; Carbohydrates I

  48. Properties • Enantiomers have identical physical properties (MP,BP, solubility, surface tension…) except when they interact with other chiral molecules • (Note!: water isn’t chiral!) • Stereoisomers that aren’t enantiomers can have different properties; therefore, they’re often given different names Biochem: Functions II; Carbohydrates I

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