1. Bioenergetics The tiny hummingbirds can store enough fuel to fly a distance of 500 miles without resting. This achievement is possible because of the ability to convert fuels into the cellular energy currency, ATP.

2. Metabolism - the entire network of chemical reactions carried out by living cells. Metabolism also includes coordination, regulation and energy requirement. Metabolites - small molecule intermediates in the degradation and synthesis of polymers Most organism use the same general pathway for extraction and utilization of energy. All living organisms are divided into two major classes: Autotrophs – can use atmospheric carbon dioxide as a sole source of carbon for the synthesis of macromolecules. Autotrophs use the sun energy for biosynthetic purposes. Heterotrophs – obtain energy by ingesting complex carbon- containing compounds. Heterotrophs are divided into aerobs and anaerobs.

3. Common features of organisms 1. Organisms or cells maintain specific internal concentrations of inorganic ions, metabolites and enzymes 2. Organisms extract energy from external sources to drive energy-consuming reactions 3. Organisms grow and reproduce according to instructions encoded in the genetic material 4. Organisms respond to environmental influences 5. Cells are not static, and cell components are continually synthesized and degraded (i.e. undergo turnover)

4. (a) Linear (b) Cyclic (c) Spiral pathway (fatty acid biosynthesis) A sequence of reactions that has a specific purpose (for instance: degradation of glucose, synthesis of fatty acids) is called metabolic pathway. Metabolic pathway may be:

5. Catabolic reactions - degrade molecules to create smaller molecules and energy Anabolic reactions - synthesize molecules for cell maintenance, growth and reproduction Metabolic pathways can be grouped into two paths – catabolism and anabolism Catabolism is characterized by oxidation reactions and by release of free energy which is transformed to ATP. Anabolism is characterized by reduction reactions and by utilization of energy accumulated in ATP molecules. Catabolism and anabolism are tightly linked together by their coordinated energy requirements: catabolic processes release the energy from food and collect it in the ATP; anabolic processes use the free energy stored in ATP to perform work.

6. Anabolism and catabolism are coupled by energy

7. Multiple-step pathways permit control of energy input and output Catabolic multi-step pathways provide energy in smaller stepwise amounts Each enzyme in a multi- step pathway usually catalyzes only one single step in the pathway Control points occur in multistep pathways Metabolism Proceeds by Discrete Steps Single-step vs multi-step pathways A multistep enzyme pathway releases energy in smaller amounts that can be used by the cell

8. Metabolism is highly regulated to permit organisms to respond to changing conditions Most pathways are irreversible Flux - flow of material through a metabolic pathway which depends upon: (1) Supply of substrates (2) Removal of products (3) Pathway enzyme activities Metabolic Pathways Are Regulated

9. Levels of Metabolism Regulation 1.Nervous system. 2.Endocrine system. 3.Interaction between organs. 4.Cell (membrane) level. 5.Molecular level

10. Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Feedback inhibition Metabolite early in the pathway activates an enzyme further down the pathway Feed-forward activation

11. Interconvertible enzyme activity can be rapidly and reversibly altered by covalent modification Protein kinases phosphorylate enzymes (+ ATP) Protein phosphatases remove phosphoryl groups Covalent modification for enzyme regulation

12. Regulatory role of a protein kinase, amplification by a signaling cascade The initial signal may be amplified by the “cascade” nature of this signaling

13. Stages of metabolism Catabolism Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA) Stage III. Acetyl CoA is oxidized in citric acid cycle to CO 2 and water. As result reduced cofactor, NADH 2 and FADH 2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

14. Glycerol Catabolism

15. Catabolism is characterized by convergence of three major routs toward a final common pathway. Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle. Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence. Monosaccharide synthesis begin with CO 2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl CoA. On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.

16. Compartmentation of metabolic processes permits: - separate pools of metabolites within a cell - simultaneous operation of opposing metabolic paths - high local concentrations of metabolites Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria) Compartmentation of Metabolic Processes in Cell

17. Compartmentation of metabolic processes

18. The chemistry of metabolism There are about 3000 reactions in human cell. All these reactions are divided into six categories: 1.Oxidation-reduction reactions 2.Group transfer reactions 3.Hydrolysis reactions 4.Nonhydrolytic cleavage reactions 5.Isomerization and rearrangement reactions 6.Bond formation reactions using energy from ATP

19. 1. Oxidation-reduction reactions -oxidases - peroxidases - dehydrogenases -oxigenases Oxidation-reduction reactions are those in which electrons are transferred from one molecule or atom to another Enzymes: oxidoreductases Coenzymes: NAD +, NADP +, FAD +, FMN + Example:

20. 2. Group transfer reactions Transfer of a chemical functional group from one molecule to another (intermolecular) or group transfer within a single molecule (intramolecular) Enzymes: transferases Examples: Phosphorylation Acylation Glycosylation

21. 3. Hydrolysis reactions Water is used to split the single molecule into two molecules - esterases - peptidases - glycosidases Enzymes: hydrolases Example:

22. 4. Nonhydrolytic cleavage reactions Split or lysis of a substrate, generating a double bond in a nonhydrolytic (without water), nonoxidative elimination Example: Enzymes: lyases

23. 5. Isomerization and rearrangement reactions Two kinds of chemical transformation: 1. Intramolecular hydrogen atom shifts changing the location of a double bond. 2. Intramolecular rearrangment of functional groups. Enzymes: isomerases Example:

24. Ligation, or joining of two substrates Require chemical energy (e.g. ATP) 6. Bond formation reactions using energy from ATP Enzymes: ligases (synthetases)

25. Add labeled substrate to tissues, cells, and follow emergence of intermediates. Use sensitive isotopic tracers ( 3 H, 14 C etc) Verify pathway steps in vitro by using isolated enzymes and substrates Study of the mutations in genes associated with the production of defective enzymes Use metabolic inhibitors to identify individual steps and sequence of enzymes in a pathway Experimental Methods for Studying Metabolism

26. OXIDATIVE DECARBOXYLATION OF PYRUVATE Matrix of the mitochondria contains pyruvate dehydrogenase complex

27. The fate of glucose molecule in the cell Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A

28. Only about 7 % of the total potential energy present in glucose is released in glycolysis. Glycolysis is preliminary phase, preparing glucose for entry into aerobic metabolism. Pyruvate formed in the aerobic conditions undergoes conversion to acetyl CoA by pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle. Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria. OXIDATIVE DECARBOXYLATION OF PYRUVATE

29. Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H + and exchange (antiport) for OH -. Entry of Pyruvate into the Mitochondrion Pyruvate freely diffuses through the outer membrane of mitochon- dria through the channels formed by transmembrane proteins porins.

30. Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins. Conversion of Pyruvate to Acetyl CoA Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons. Electron micrograph of the pyruvate dehydrogenase complex from E. coli.

31. Enzymes: E1 = pyruvate dehydrogenase E2 = dihydrolipoyl acetyltransferase E3 = dihydrolipoyl dehydrogenase Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-CoA, FAD+, NAD+. TPP is a prosthetic group of E1; lipoamide is a prosthetic group of E2; and FAD is a prosthetic group of E3. The building block of TPP is vitamin B 1 (thiamin); NAD – vitamin B 5 (nicotinamide); FAD – vitamin B 2 (riboflavin), HS-CoA – vitamin B 3 (pantothenic acid), lipoamide – lipoic acid

32. Overall reaction of pyruvate dehydrogenase complex Pyruvate dehydrogenase complex is a classic example of multienzyme complex The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

33. The Citric Acid Cycle Aerobic cells use a metabolic wheel – the citric acid cycle – to generate energy by acetyl CoA oxidation

34. Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A Fatty Acids Amino Acids The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A.

35. Names: The Citric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle In eukaryotes the reactions of the citric acid cycle take place inside mitochondria Physiology or Medicine. Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.

36. An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate. An isomer of citrate is oxidatively decarboxylated and five-carbon  - ketoglutarate is formed.  -ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate. Oxaloacetate is then regenerated from succinate. Two carbon atoms (acetyl CoA) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (six electrons) are transferred to three molecules of NAD +, one pair of hydrogen atoms (two electrons) is transferred to one molecule of FAD. The function of the citric acid cycle is the harvesting of high- energy electrons from acetyl CoA.

37. 1. Citrate Synthase Citrate formed from acetyl CoA and oxaloacetate Only cycle reaction with C-C bond formation Addition of C 2 unit (acetyl) to the keto double bond of C 4 acid, oxaloacetate, to produce C 6 compound, citrate citrate synthase

38. 2. Aconitase Elimination of H 2 O from citrate to form C=C bond of cis-aconitate Stereospecific addition of H 2 O to cis-aconitate to form isocitrate aconitase

39. 3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD + to form NADH Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

40. 4. The  -Ketoglutarate Dehydrogenase Complex Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E 1 - a-ketoglutarate dehydrogenase (with TPP) E 2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E 3 - dihydrolipoyl dehydrogenase (with FAD)  -ketoglutarate dehydrogenase

41. 5. Succinyl-CoA Synthetase Free energy in thioester bond of succinyl CoA is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) Substrate level phosphorylation reaction HS- + GTP + ADP GDP + ATP Succinyl-CoA Synthetase

42. Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Embedded in the inner mitochondrial membrane Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain Dehydrogenation is stereospecific; only the trans isomer is formed 6. The Succinate Dehydrogenase Complex Succinate Dehydrogenase

43. 7. Fumarase Stereospecific trans addition of water to the double bond of fumarate to form L-malate Only the L isomer of malate is formed Fumarase

44. 8. Malate Dehydrogenase Malate Dehydrogenase Malate is oxidized to form oxaloacetate.

45. Stoichiometry of the Citric Acid Cycle  Two carbon atoms enter the cycle in the form of acetyl CoA.  Two carbon atoms leave the cycle in the form of CO 2.  Four pairs of hydrogen atoms leave the cycle in four oxidation reactions (three molecules of NAD + one molecule of FAD are reduced).  One molecule of GTP, is formed.  Two molecules of water are consumed.  9 ATP (2.5 ATP per NADH, and 1.5 ATP per FADH 2 ) are produced during oxidative phosphorylation.  1 ATP is directly formed in the citric acid cycle.  1 acetyl CoA generates approximately 10 molecules of ATP.

46. Integration of metabolism. The citric acid cycle is amphibolic (both catabolic and anabolic). Functions of the Citric Acid Cycle The cycle is involved in the aerobic catabolism of carbohydrates, lipids and amino acids. Intermediates of the cycle are starting points for many anabolic reactions. Yields energy in the form of GTP (ATP). Yields reducing power in the form of NADH 2 and FADH 2.

47. Regulation of the Citric Acid Cycle Pathway controlled by: (1) Allosteric modulators (2) Covalent modification of cycle enzymes (3) Supply of acetyl CoA (pyruvate dehydrogenase complex) Three enzymes have regulatory properties - citrate synthase (is allosterically inhibited by NADH, ATP, succinyl CoA, citrate – feedback inhibition) - isocitrate dehydrogenase (allosteric effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently modified by kinase/phosphatase) -  -ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl CoA and NADH

48. NADH, ATP, succinyl CoA, citrate - Regulation of the citric acid cycle

49. Krebs Cycle is a Source of Biosynthetic Precursors Phosphoenol- pyruvate Glucose The citric acid cycle provides intermediates for biosyntheses