1. 11/17/2010Motors II; Metabolism I Motors II; General Metabolism I Andy Howard Introductory Biochemistry, fall 2010 17 November 2010

2. 11/17/2010Motors II; Metabolism I Page 2 of 40 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 … but first, we need to explain the role of Ca 2+ in muscle contraction

3. 11/17/2010Motors II; Metabolism I Page 3 of 40 What we’ll discuss Muscle Calcium Ca 2+ receptors Troponin C and I Smooth muscle Metabolism Definitions Pathways Metabolism, continued Control Feedback Flux Phosphorylation Other PTMs Evolution

4. 11/17/2010Motors II; Metabolism I Page 4 of 40 Muscle Contraction Is Regulated by Ca 2+ Ca 2+ Channels and Pumps Release of Ca 2+ from the SR triggers contraction Reuptake of Ca 2+ into SR relaxes muscle So how is calcium released in response to nerve impulses? Answer has come from studies of antagonist molecules that block Ca 2+ channel activity

5. 11/17/2010Motors II; Metabolism I Page 5 of 40 Ca 2+ triggers contraction Release of Ca 2+ through voltage- or Ca 2+ -sensitive channel activates contraction Pumps induce relaxation

6. 11/17/2010Motors II; Metabolism I Page 6 of 40 Dihydropyridine Receptor In t-tubules of heart and skeletal muscle Nifedipine and other DHP-like molecules bind to the "DHP receptor" in t-tubules In heart, DHP receptor is a voltage-gated Ca 2+ channel In skeletal muscle, DHP receptor is apparently a voltage-sensing protein and probably undergoes voltage-dependent conformational changes

7. 11/17/2010Motors II; Metabolism I Page 7 of 40 Ryanodine Receptor The "foot structure" in terminal cisternae of SR Foot structure is a Ca 2+ channel of unusual design Conformation change or Ca 2+ -channel activity of DHP receptor apparently gates the ryanodine receptor, opening and closing Ca 2+ channels Many details are yet to be elucidated!

8. 11/17/2010Motors II; Metabolism I Page 8 of 40 Ryanodine Receptor Courtesy BBRI

9. 11/17/2010Motors II; Metabolism I Page 9 of 40 Muscle Contraction Is Regulated by Ca 2+ Tropomyosin and troponins mediate the effects of Ca 2+ See Figure 16.24 In absence of Ca 2+, TnI binds to actin to keep myosin off TnI and TnT interact with tropomyosin to keep tropomyosin away from the groove between adjacent actins But Ca 2+ binding changes all this!

10. 11/17/2010Motors II; Metabolism I Page 10 of 40 Ca 2+ Turns on Contraction Binding of Ca 2+ to TnC increases binding of TnC to TnI, simultaneously decreasing the interaction of TnI with actin This allows tropomyosin to slide down into the actin groove, exposing myosin-binding sites on actin and initiating contraction Since troponin complex interacts only with every 7th actin, the conformational changes must be cooperative

11. 11/17/2010Motors II; Metabolism I Page 11 of 40 Thin & thick filaments Changes that happen when Ca 2+ binds to troponin C Fig. 16.24

12. 11/17/2010Motors II; Metabolism I Page 12 of 40 Binding of Ca 2+ to Troponin C Four sites for Ca 2+ on TnC - I, II, III and IV Sites I & II are N-terminal; III and IV on C term Sites III and IV usually have Ca 2+ bound Sites I and II are empty in resting state Rise of Ca 2+ levels fills sites I and II Conformation change facilitates binding of TnC to TnI

13. 11/17/2010Motors II; Metabolism I Page 13 of 40 2 views of troponin C Ribbon Molecular graphic Fig. 16.25

14. 11/17/2010Motors II; Metabolism I Page 14 of 40 Smooth Muscle Contraction No troponin complex in smooth muscle In smooth muscle, Ca 2+ activates myosin light chain kinase (MLCK) which phosphorylates LC2, the regulatory light chain of myosin Ca 2+ effect is via calmodulin - a cousin of Troponin C

15. 11/17/2010Motors II; Metabolism I Page 15 of 40 Effect of hormones on smooth muscle Hormones regulate contraction - epinephrine, a smooth muscle relaxer, activates adenylyl cyclase, making cAMP, which activates protein kinase, which phosphorylates MLCK, inactivating MLCK and relaxing muscle

16. 11/17/2010Motors II; Metabolism I Page 16 of 40 Smooth Muscle Effectors Useful drugs Epinephrine (as Primatene) is an over-the- counter asthma drug, but it acts on heart as well as on lungs - a possible problem! Albuterol is a more selective smooth muscle relaxer and acts more on lungs than heart Albuterol is used to prevent premature labor Oxytocin (pitocin) stimulates contraction of uterine smooth muscle, inducing labor

17. 11/17/2010Motors II; Metabolism I Page 17 of 40 Oxytocin structure P.532

18. 11/17/2010Motors II; Metabolism I Page 18 of 40 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!

19. 11/17/2010Motors II; Metabolism I Page 19 of 40 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?

20. 11/17/2010Motors II; Metabolism I Page 20 of 40 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

21. 11/17/2010Motors II; Metabolism I Page 21 of 40 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

22. 11/17/2010Motors II; Metabolism I Page 22 of 40 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

23. 11/17/2010Motors II; Metabolism I Page 23 of 40 Metabolism and energy

24. 11/17/2010Motors II; Metabolism I Page 24 of 40 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

25. 11/17/2010Motors II; Metabolism I Page 25 of 40 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,...

26. 11/17/2010Motors II; Metabolism I Page 26 of 40 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

27. 11/17/2010Motors II; Metabolism I Page 27 of 40 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

28. 11/17/2010Motors II; Metabolism I Page 28 of 40 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

29. 11/17/2010Motors II; Metabolism I Page 29 of 40 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

30. 11/17/2010Motors II; Metabolism I Page 30 of 40 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

31. 11/17/2010Motors II; Metabolism I Page 31 of 40 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.

32. 11/17/2010Motors II; Metabolism I Page 32 of 40 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

33. 11/17/2010Motors II; Metabolism I Page 33 of 40 Differences between catabolic and anabolic pathways Often they share many reactions, notably the ones that are nearly isoergic (  G o ~ 0) Reactions with  G o < -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

34. 11/17/2010Motors II; Metabolism I Page 34 of 40 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

35. 11/17/2010Motors II; Metabolism I Page 35 of 40 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

36. 11/17/2010Motors II; Metabolism I Page 36 of 40 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 (2.5 ATP/NAD) in oxidative phosphorylation Image courtesy Michigan Tech Biological Sciences

37. 11/17/2010Motors II; Metabolism I Page 37 of 40 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

38. 11/17/2010Motors II; Metabolism I Page 38 of 40 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

39. 11/17/2010Motors II; Metabolism I Page 39 of 40 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

40. 11/17/2010Motors II; Metabolism I Page 40 of 40 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.