1. 11/24/2009Biochemistry: Metabolism I General Metabolism I Andy Howard Introductory Biochemistry 24 November 2009

2. 11/24/2009Biochemistry: Metabolism I Page 2 of 75 Metabolism: the core of biochem All of biology 402 will concern itself with the specific pathways of metabolism Our purpose here is to arm you with the necessary weaponry

3. 11/24/2009Biochemistry: Metabolism I Page 3 of 75 What we’ll discuss Metabolism Definitions Pathways Control Feedback Phosphorylation Thermodynamics Kinetics Cofactors Tightly-bound metal ions as cofactors Activator ions as cofactors Cosubstrates Prosthetic groups

4. 11/24/2009Biochemistry: Metabolism I Page 4 of 75 Metabolism Almost ready to start the specifics (chapter 18) Define it! Metabolism is the network of chemical reactions that occur in biological systems, including the ways in which they are controlled. So it covers most of what we do here!

5. 11/24/2009Biochemistry: Metabolism I Page 5 of 75 Intermediary Metabolism Metabolism involving small molecules Describing it this way is a matter of perspective: Do the small molecules exist to give the proteins something to do, or do the proteins exist to get the metabolites interconverted?

6. 11/24/2009Biochemistry: Metabolism I Page 6 of 75 How similar are pathways in various organisms? Enormous degree of similarity in the general metabolic approaches all the way from E.coli to elephants Glycolysis arose prior to oxygenation of the atmosphere This is considered strong evidence that all living organisms are derived from a common ancestor

7. 11/24/2009Biochemistry: Metabolism I Page 7 of 75 Anabolism and catabolism Anabolism: synthesis of complex molecules from simpler ones Generally energy-requiring Involved in making small molecules and macromolecules Catabolism: degradation of large molecules into simpler ones Generally energy-yielding All the sources had to come from somewhere

8. 11/24/2009Biochemistry: Metabolism I Page 8 of 75 Common metabolic themes Maintenance of internal concentrations of ions, metabolites, & (? enzymes) Extraction of energy from external sources Pathways specified genetically Organisms & cells interact with their environment Constant degradation & synthesis of metabolites and macromolecules to produce steady state

9. 11/24/2009Biochemistry: Metabolism I Page 9 of 75 Metabolism and energy

10. 11/24/2009Biochemistry: Metabolism I Page 10 of 75 Metabolic classifications Carbon sources Autotrophs vs. heterotrophs Atmospheric CO 2 as a C source vs. otherwise-derived C sources Energy sources Phototrophs vs. chemotrophs (Sun)light as source of energy vs. reduced organic compounds as a source of energy

11. 11/24/2009Biochemistry: Metabolism I Page 11 of 75 Fourway divisions (table 17.2) Energy/CarbonPhototrophs: Energy from light Chemotrophs: Energy from reduced organic molecules Autotrophs: Carbon from atmospheric CO 2 Photoautotrophs: Green plants, cyanobacteria, … Chemoautotrophs: Nitrifying bacteria, H, S, Fe bacteria Heterotrophs: Carbon from other [organic] sources Photoheterotrophs: Nonsulfur purple bacteria Chemoheterotrophs: Animals, many microorganisms,...

12. 11/24/2009Biochemistry: Metabolism I Page 12 of 75 Another distinction: the organism and oxygen Aerobes: use O 2 as the ultimate electron acceptor in oxidation-reduction reactions Anaerobes: don’t depend on O 2 Obligate: poisoned by O 2 Facultative: can switch hit

13. 11/24/2009Biochemistry: Metabolism I Page 13 of 75 Flow of energy Sun is ultimate source of energy Photoautotrophs drive synthesis of [reduced] organic compounds from atmospheric CO 2 and water Chemoheterotrophs use those compounds as energy sources & carbon; CO 2 returned to atmosphere

14. 11/24/2009Biochemistry: Metabolism I Page 14 of 75 How to anabolism & catabolism interact? Sometimes anabolism & catabolism occur simultaneously. How do cells avoid futile cycling? Just-in-time metabolism Compartmentalization: Anabolism often cytosolic Catabolism often mitochondrial

15. 11/24/2009Biochemistry: Metabolism I Page 15 of 75 Pathway A sequence of reactions such that the product of one is the substrate for the next Similar to an organic synthesis scheme (but with better yields!) May be: Unbranched Branched Circular

16. 11/24/2009Biochemistry: Metabolism I Page 16 of 75 Catabolism stages Stage 1: big nutrient macromolecules hydrolyzed into their building blocks Stage 2: Building blocks degraded into limited set of simpler intermediates, notably acetyl CoA Stage 3: Simple intermediates are fed to TCA cycle and oxidative phosphorylation

17. 11/24/2009Biochemistry: Metabolism I Page 17 of 75 Anabolism stages Short list of simple precursors These are elaborated in characteristic ways to build monomers e.g.: transamination of  -ketoacids to make  -amino acids Those are then polymerized to form proteins, polysaccharides, polynucleotides, etc.

18. 11/24/2009Biochemistry: Metabolism I Page 18 of 75 Some intermediates play two roles Some metabolites play roles in both kinds of pathways We describe them as amphibolic Just recall that: catabolism is many down to few, anabolism is few up to many

19. 11/24/2009Biochemistry: Metabolism I Page 19 of 75 Differences between catabolic and anabolic pathways Often they share many reactions, notably the ones that are nearly isoergic (  G ~ 0) Reactions with  G < -20 kJ mol -1 are not reversible as is Those must be replaced by (de)coupled reactions so that the oppositely-signed reactions aren’t unfeasible

20. 11/24/2009Biochemistry: Metabolism I Page 20 of 75 Other differences involve regulation Generally control mechanisms influence catalysis in both directions Therefore a controlling influence (e.g. an allosteric effector) will up- or down-regulate both directions If that’s not what the cell needs, it will need asymmetric pathways or pathways involving different enzymes in the two directions

21. 11/24/2009Biochemistry: Metabolism I Page 21 of 75 ATP’s role We’ve discussed its significance as an energy currency It’s one of two energy-rich products of the conversion of light energy into chemical energy in phototrophs ATP then provides drivers for almost everything else other than redox

22. 11/24/2009Biochemistry: Metabolism I Page 22 of 75 NAD’s role NAD acts as as an electron acceptor via net transfer of hydride ions, H: -, in catabolic reactions Reduced substrates get oxidized in the process, and their reducing power ends up in NADH Energy implied by that is used to make ATP (3.5 ATP/NAD) in oxidative phosphorylation Image courtesy Michigan Tech Biological Sciences

23. 11/24/2009Biochemistry: Metabolism I Page 23 of 75 NADPH’s role Involved in anabolic redox reactions Reducing power in NADPH  NADP used to reduce some organic molecule Involves hydride transfers again NADPH regenerated in phototrophs via light-dependent reactions that pull electrons from water

24. 11/24/2009Biochemistry: Metabolism I Page 24 of 75 How do we study pathways? Inhibitor studies Mutagenesis Isotopic traces (radio- or not) NMR Disruption of cells to examine which reactions take place in which organelle

25. 11/24/2009Biochemistry: Metabolism I Page 25 of 75 Why multistep pathways? Limited reaction specificity of enzymes Control of energy input and output: Break big inputs into ATP-sized inputs Break energy output into pieces that can be readily used elsewhere

26. 11/24/2009Biochemistry: Metabolism I Page 26 of 75 iClicker quiz question 1 A reaction A+B  C+D proceeds from left to right in the cytosol and from right to left in the mitochondrion. As written, it is probably (a) a catabolic reaction (b) an anabolic reaction (c) an amphibolic reaction (d) we don’t have enough information to answer.

27. 11/24/2009Biochemistry: Metabolism I Page 27 of 75 iClicker quiz question 2 An asymmetry between stage 1 of catabolism (C1) and the final stage of anabolism (A3) is (a) A3 always requires light energy; C1 doesn’t (b) A3 never produces nucleotides; C1 can involve nucleotide breakdown (c) A3 adds one building block at a time to the end of the growing polymer; C1 can involve hydrolysis in the middle of the polymer (d) There are no asymmetries between A3 and C1

28. 11/24/2009Biochemistry: Metabolism I Page 28 of 75 iClicker quiz question 3 Could dAMP, derived from degradation of DNA, serve as a building block to make NADP? (a) Yes. (b) Probably not: the energetics wouldn’t allow it. (c) Probably not: the missing 2’-OH would make it difficult to build NADP (d) No: dAMP is never present in the cell

29. 11/24/2009Biochemistry: Metabolism I Page 29 of 75 Regulation Organisms respond to change Fastest: small ions move in msec Metabolites: 0.1-5 sec Enzymes: minutes to days Flow of metabolites is flux: steady state is like a leaky bucket Addition of new material replaces the material that leaks out the bottom

30. 11/24/2009Biochemistry: Metabolism I Page 30 of 75 Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial

31. 11/24/2009Biochemistry: Metabolism I Page 31 of 75 Feedback and Feed-forward Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway

32. 11/24/2009Biochemistry: Metabolism I Page 32 of 75 Feedback realities Control usually exerted at first committed step (i.e., the first reaction that is unique to the pathway) Controlling element is usually the last element in the path Often the controlled reaction has a large negative  G o ’.

33. 11/24/2009Biochemistry: Metabolism I Page 33 of 75 Feed-forward Early metabolite activates a reaction farther down the pathway Has the potential for instabilities, just as in electrical feed-forward Usually modulated by feedback

34. 11/24/2009Biochemistry: Metabolism I Page 34 of 75 Activation and inactivation by post-translational modification Most common: covalent phosphorylation of protein usually S, T, Y, sometimes H Kinases add phosphate Protein-OH + ATP  Protein-O-P + ADP … ATP is source of energy and P i Phosphatases hydrolyze phosphoester: Protein-O-P +H 2 O  Protein-OH + P i … no external energy source required

35. 11/24/2009Biochemistry: Metabolism I Page 35 of 75 Phosphorylation’s effects Phosphorylation of an enzyme can either activate it or deactivate it Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated Example: glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme

36. 11/24/2009Biochemistry: Metabolism I Page 36 of 75 Glycogen phosphorylase Reaction: extracts 1 glucose unit from non-reducing end of glycogen & phosphorylates it: (glycogen) n + P i  (glycogen) n-1 + glucose-1-P Activated by phosphorylation via phosphorylase kinase Deactivated by dephosphorylation by phosphorylase phosphatase

37. 11/24/2009Biochemistry: Metabolism I Page 37 of 75 Amplification Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins Thus a single activation event at the kinase level can trigger many events at the target level

38. 11/24/2009Biochemistry: Metabolism I Page 38 of 75 Other PTMs Are there other reversible post- translational modifications that regulate enzyme activity? Yes: Adenylation of Y ADP-ribosylation of R Uridylylation of Y Oxidation of cysteine pairs to cystine Cis-trans isomerization of prolines

39. 11/24/2009Biochemistry: Metabolism I Page 39 of 75 Evolution of Pathways: How have new pathways evolved? Add a step to an existing pathway Evolve a branch on an existing pathway Backward evolution Duplication of existing pathway to create related reactions Reversing an entire pathway

40. 11/24/2009Biochemistry: Metabolism I Page 40 of 75 Adding a step A  B  C  D  E  P When the organism makes lots of E, there’s good reason to evolve an enzyme E 5 to make P from E. This is how asn and gln pathways (from asp & glu) work E1E1 E2E2 E3E3 E4E4 E5E5 Original pathway

41. 11/24/2009Biochemistry: Metabolism I Page 41 of 75 Evolving a branch Original pathway: D A  B  C X Fully evolved pathway: D A  B  C X E1E1 E2E2 E3E3 E 3a E 3b

42. 11/24/2009Biochemistry: Metabolism I Page 42 of 75 Backward evolution Original system has lots of E  P E gets depleted over time; need to make it from D, so we evolve enzyme E4 to do that. Then D gets depleted; need to make it from C, so we evolve E3 to do that And so on

43. 11/24/2009Biochemistry: Metabolism I Page 43 of 75 Duplicated pathways Homologous enzymes catalyze related reactions; this is how trp and his biosynthesis enzymes seem to have evolved Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination)

44. 11/24/2009Biochemistry: Metabolism I Page 44 of 75 Reversing a pathway We’d like to think that lots of pathways are fully reversible Usually at least one step in any pathway is irreversible (  G o ’ < -15 kJ mol -1 ) Say C  D is irreversible so E 3 only works in the forward direction Then D + ATP  C + ADP + P i allows us to reverse that one step with help The other steps can be in common This is how glycolysis evolved from gluconeogenesis

45. 11/24/2009Biochemistry: Metabolism I Page 45 of 75 Many cofactors are derived from vitamins We justify lumping these two topics together because many cofactors are vitamins or are metabolites of vitamins.

46. 11/24/2009Biochemistry: Metabolism I Page 46 of 75 Family tree of cofactors Cofactors, coenzymes, essential ions, cosubstrates, prosthetic groups: Cofactors (apoenzyme + cofactor  holoenzyme) Essential ions Coenzymes Activator ions (loosely bound) Ions in metalloenzymes Prosthetic groups (tightly bound) Cosubstrates (loosely bound)

47. 11/24/2009Biochemistry: Metabolism I Page 47 of 75 Metal-activated enzymes Absolute requirements for mobile ions Often require K +, Ca 2+, Mg 2+ Example: Kinases: Mg-ATP complex Metalloenzymes: firmly bound metal ions in active site Usually divalent or more Sometimes 1e - redox changes in metal

48. 11/24/2009Biochemistry: Metabolism I Page 48 of 75 Coenzymes Organic moeities that enable enzymes to perform their function: they supply functionalities not available from amino acid side chains Cosubstrates Enter reaction, get altered, leave Repeated recycling within cell or organelle Prosthetic groups Remain bound to enzyme throughout Change during one phase of reaction, eventually get restored to starting state

49. 11/24/2009Biochemistry: Metabolism I Page 49 of 75 Major cosubstrates Facilitate group transfers, mostly small groups Oxidation-reduction participants CosubstrateSourceFunction ATPTransfer P,Nucleotide S-adenosylMetMethyl transfer UDP-glucoseGlycosyl transfer NAD,NADPNiacin2-electron redox Coenzyme APantothenateAcyl transfer TetrahydrofolateFolate1Carbon transfer UbiquinoneLipid-soluble e - carrier

50. 11/24/2009Biochemistry: Metabolism I Page 50 of 75 Major prosthetic groups Transfer of larger groups One- or two-electron redox changes Prosth.gp.SourceFunction FMN, FADRiboflavin1e - and 2e - redox transfers TPPThiamine2-Carbon transfers with C=O PLPPyridoxineAmino acid group transfers BiotinBiotinCarboxylation, COO - transfer Adenosyl-CobalaminIntramolec. rearrangements cobalamin MeCobal.CobalaminMethyl-group transfers LipoamideTransfer from TPP RetinalVitamin AVision Vitamin KVitamin KCarboxylation of glu residues

51. 11/24/2009Biochemistry: Metabolism I Page 51 of 75 Adenosine triphosphate Synthesizable in liver (chapter 18) Building block for RNA Participates in phosphoryl-group transfer in kinases Source of other coenzymes

52. 11/24/2009Biochemistry: Metabolism I Page 52 of 75 S-adenosylmethionine Made from methionine and adenosine Sulfonium group is highly reactive: can donate methyl groups Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University

53. 11/24/2009Biochemistry: Metabolism I Page 53 of 75 UDP-glucose Most common donor of glucose Formed via: Glucose-1P + UTP  UDP-glucose + PP i Reaction driven to right by PP i hydrolysis Structure courtesy of UIC Pharmacy Program

54. 11/24/2009Biochemistry: Metabolism I Page 54 of 75 NAD + and NADP + Net charge isn’t really >0 ; the + is just a reminder that the nicotinamide ring is positively charged Most important cosubstrates in oxidation- reduction reactions in aerobic organisms Structure courtesy of Sergio Marchesini, U. Brescia

55. 11/24/2009Biochemistry: Metabolism I Page 55 of 75 Differences between them The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety The functional difference is that NAD is usually associated with catabolic reactions and NADP is usually associated with anabolic reactions Therefore often NAD + and NADPH are reactants and NADH and NADP + are products Exceptions: photosynthesis and ETC!

56. 11/24/2009Biochemistry: Metabolism I Page 56 of 75 How do we get back to the starting point? NADH is often oxidized back to NAD + as part of the electron-transport chain Imbalances can be addressed via NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase

57. 11/24/2009Biochemistry: Metabolism I Page 57 of 75 iClicker quiz question 4 Based on what you have learned, would you expect glycogen synthase to be activated or inhibited by phosphorylation? (a) activated (b) inhibited (c) neither (d) insufficient information to tell

58. 11/24/2009Biochemistry: Metabolism I Page 58 of 75 iClicker quiz question 5 What would you expect to be the phosphate donor in the NAD kinase reaction? (a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate

59. 11/24/2009Biochemistry: Metabolism I Page 59 of 75 Reduced forms of NAD(P) Reduction occurs on the nicotinamide ring Ring is no longer net- positive Ring is still planar but the two hydrogens on the para carbon are not

60. 11/24/2009Biochemistry: Metabolism I Page 60 of 75 FAD and FMN Flavin group based on riboflavin Alternate participants in redox reactions Prosthetic groups: tightly but noncovalently bound to their enzymes That protects against wasteful reoxidation of reduced forms FADH 2 is weaker reducing agent than NADH These are capable of one-electron oxidations and reductions

61. 11/24/2009Biochemistry: Metabolism I Page 61 of 75 FAD and FMN structures FAD has an AMP attached P to P Structure courtesy Paisley University

62. 11/24/2009Biochemistry: Metabolism I Page 62 of 75 Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University FMN/FAD redox forms Two-electron version: H + + :H - transferred

63. 11/24/2009Biochemistry: Metabolism I Page 63 of 75 Coenzyme A Reactive portion is free sulfhydryl at one end of the molecule Can form thioester with acetate, etc. Pantoate +  -alanine = pantothenate Structure courtesy of MPB project, George Washington University (ADP-3’P) (Pantoate)  -alanine)  2-mercapto- ethylamine)

64. 11/24/2009Biochemistry: Metabolism I Page 64 of 75 Thiamine Pyrophosphate Based on thiamine, vitamin B1 Carboxylases and oxidative decarboxylases use this coenzyme So do transketolases (move 2 carbons at a time between sugars with keto groups) Thiazolium ring is reactive center: pK a drops from 15 in H 2 O to 6 in enzyme

65. 11/24/2009Biochemistry: Metabolism I Page 65 of 75 TPP reactions Diagram courtesy of Oklahoma State U. Biochemistry program pyrimidine thiazolium

66. 11/24/2009Biochemistry: Metabolism I Page 66 of 75 Pyridoxal phosphate PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations Carbonyl group of PLP bound as a Schiff base (imine) to  -amino group of lysine at active site First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine

67. 11/24/2009Biochemistry: Metabolism I Page 67 of 75 Biotin Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl groups … and in ATP-dependent carboxylations

68. 11/24/2009Biochemistry: Metabolism I Page 68 of 75 Biotin reactivity Covalently bound to active-site lysines to form species called biocytin Pyruvate carboxylase is characteristic reaction: Diagram courtesy University of Virginia Biochemistry

69. 11/24/2009Biochemistry: Metabolism I Page 69 of 75 Tetrahydrofolate Primary donor of one-carbon units (formyl, methylene, methyl) Supplies methyl group for thymidylate Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells Trimethoprim as antibacterial: Bacterial DHFR is somewhat different from eucaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate

70. 11/24/2009Biochemistry: Metabolism I Page 70 of 75 THF structure and function Figure courtesy horticulture program, Purdue

71. 11/24/2009Biochemistry: Metabolism I Page 71 of 75 Cobalamin Largest B vitamin Structure related to heme but missing one carbon in ring structure Cobalt bound in core of ring system Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation

72. 11/24/2009Biochemistry: Metabolism I Page 72 of 75 Adenosyl- Cobalamin Diagram courtesy of Swiss Food News “Missing” carbon Reactive Co-C bond

73. 11/24/2009Biochemistry: Metabolism I Page 73 of 75 Lipoamide Protein-bound form of lipoic acid Contains five-membered disulfide ring Covalently bound via amide to protein lysine sidechain Involved in swinging arm between active sites in multienzyme complexes Disulfides break periodically Example: pyruvate dehydrogenase complex

74. 11/24/2009Biochemistry: Metabolism I Page 74 of 75 Lipoamide 2e - reduction Cf. Scheme 7.6: thioester starting point Fig. Courtesy Biochem and Biophysics program, Rensselaer

75. 11/24/2009Biochemistry: Metabolism I Page 75 of 75 iClicker quiz question 6 Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate  aspartate +  -ketoglutarate? (a) ascorbate (b) PLP ( c) thiamine pyrophosphate (d) NAD (e) none of the above