Metabolism: basic concepts

1. Metabolism: basic concepts

2. Metabolism • Sum of the thousands of enzyme-mediated chemical reactions whereby • Energy (E) is extracted from fuels to power motion, biosynthesis and active transport • Involves many interdependent metabolic pathways (linear or cyclic) • Compounds that take part in or are formed are called metabolites • Reactions are catabolic (generation of E)or anabolic (biosynthesis)

3. Metabolism • Catabolism • Breakdown of carbon fuels (carbohydrates & fats) to CO2, H2O and E in form of ATP • Anabolism • Combining small molecules to form complex molecules e.g. proteins & fats. Requires E in form of ATP

4. O H3C-C-S-CoA ATP Stages of catabolism Stage 1: No useful energy is captured. Stage II: Acetyl CoA and a little ATP generated. Stage III: ATP generation from complete oxidation of acetyl unit of acetyl CoA. Four pairs of electrons are transferred (3 NAD+ and 1 FAD) for each acetyl group oxidised. Electrons from reduced carriers generate a proton gradient from which ATP is generated. Coenzyme A is an important coenzyme in metabolism. It is a carrier of acyl groups.

5. Free Energy • A measure of a system’s ability to do work • Gibb’s free energy, G, is defined by: G = H – TS T = ? H = ? S = ? . DG -Total free E of products – total free E of reactants • A favourable or spontaneous reaction (rxn) occurs if the change in free E (DG) is negative • Exorgenic: giving off E • Endorgenic: taking in E

6. Metabolism • A thermodynamically or energetically unfavourable rxn can be driven if a favourable rxn is coupled to it • A B + C DGo’ = +5 kcal mol-1 • B D DGo’ = -8 kcal mol-1 • A C + D DGo’ = -3 kcal mol-1 • Metabolic pathways are formed by coupling of enzyme-mediated rxns, such that overall free E change is negative

7. Definitions • Cofactor: • A nonprotein component essential for normal catalytic activity of an enzyme. • May be organic molecules (coenzymes) or inorganic ions. • May activate the enzyme by altering its shape or participate in the chemical reaction. • Coenzyme: • An organic nonprotein molecule that associates with an enzyme in catalysing biochemical reactions e.g. nicotinamide adenine dinucleotide (NAD). • usually participate in the substrate-enzyme interaction by donating or accepting chemical groups. • Many vitamins are precursors of coenzymes.

8. Coenzyme A (Co A) • A complex organic compound • Acts in conjunction with enzymes involved in biochemical rxns, notably • in the oxidation of pyruvate via the Krebs cycle and • fatty acid oxidation and synthesis. • Comprises pantothenic acid (a B vitamin), adenine (nucleotide) and a ribose-phosphate group

9. Nicotinamide adenine dinucleotide (NAD+) • Coenzyme derived from nicotinic acid (a B vitamin) • Participates in dehydrogenation rxns • Loosely bound to enzyme involved • Normally carries +ve charge and can accept 1 hydrogen atom and 2 electrons to become NADH (reduced form) • NADH (generated by oxidation of food) gives up 2 electrons (& single proton) to electron transport chain NAD+ (oxidised form) + 3 molecules ATP

10. Biotin • The coenzyme for enzymes that catalyse the incorporation of CO2 into various compounds • A vitamin in the vitamin B complex • Adequate amounts normally produced by intestinal bacteria. • Other sources include cereals, vegetables, milk and liver.

11. NH2 High E anhydride linkages N Adenine O O O CH2 N H H N N O- O P O P O P O Ribose O- O- O- H OH OH Adenosine Adenosine triphosphate (ATP): universal currency of free E in biological systems

12. Adenosine triphosphate (ATP) • Large amounts of free E released when ATP is hydrolysed • ATP + H2O ADP + Pi (orthophosphate) DGo’ =-7.3 kcal mol-1 • ADP + H2O AMP + PPi (pyrophosphate) DGo’ = -7.3 kcal mol-1 • The 2 phosphoanhydride bonds are E rich • Active form of ATP is complex with Mg2+ or Mn2+

13. Adenosine triphosphate (ATP) • ATP ADP cycle is fundamental rxn in cells which provides free E to drive rxns • ATP turnover very high; ATP molecule consumed within 1 min of formation; fairly stable in the absence of a catalyst

14. UTP, GTP & CTP • Some biosynthetic rxns are driven by the hydrolysis of analogues to ATP: • Uridine triphosphate, guanosine triphospate and cytidine triphosphate • They are energetically equivalent • Phosphoryl transfer is a common means of energy coupling

15. Phosphoryl transfer potential of ATP occupies intermediate position • Creatine phosphate + ADP + H+ ATP + creatine • ATP functions efficiently as a carrier of phosphoryl groups

16. 2 mechanisms for ATP synthesis • High phosphoryl transfer potential compounds can couple carbon oxidation (removal of electrons) to ATP synthesis (substrate-level phosphorylation). Can occur in absence of oxygen (anaerobic metabolism). • Proton gradient generated by oxidation of carbon fuels accounts for > 90% of ATP generation (oxidative phosphorylation).

17. Oxidative phosphorylation H+ H+ H+ NADH Oxidation of fuel pumps protons out H2O ADP + Pi ATP Influx of protons forms ATP H+ H+ Inner mitochondrial membrane

18. Activated carriers • ATP considered an activated carrier of phosphoryl group • Nicotinamide adenine dinucleotide (NAD+, oxidised form) • Key electron carrier for the generation of ATP • Reactive nicotinamide ring derived from niacin, vitamin B3 (oxidation of nicotine). • In substrate oxidation, NAD accepts 2 electrons and one hydrogen ion/proton (reduced) • NAD+ + 2e- + 2H+NADH+ H+ • Nicotinamide adenine dinucleotide phosphate (NADP+) • Key electron carrier in reduction biosynthesis e.g. ketone to fatty acid

19. Activated carriers • Flavin adenine dinucleotide (FAD, oxidised form). • Derived from riboflavin, vitamin B2. • An electron carrier in the generation of ATP. • In substrate oxidation, it accepts 2 electrons and 2 hydrogen ions / protons (and becomes reduced). FAD + 2H  FADH2 • Coenzyme A (CoA) • Carrier of activated acyl group. Derived from -mercaptoethylamine, panthothenate and ATP. • Acyl (e.g. acetyl) group linked to CoA by thioester bond to form acyl (acetyl) CoA. • Key in the synthesis and oxidation of fatty acids, and oxidation of pyruvate in the citric acid cycle.

20. Glycolysis

21. Metabolism • Sum of the thousands of enzyme-mediated chemical reactions whereby • Energy (E) is extracted from fuels to power motion, biosynthesis and active transport • Involves many interdependent metabolic pathways (linear or cyclic) • Glycolysis – a linear metabolic pathway

22. Polysaccharides Lipids Fatty acids Proteins Nucleotides Glucose Amino Acids 5 & 6 carbon sugars Glycerol NADPH Glucose Glycolysis Gluconeogensis Pyruvate Acetyl-CoA Citric acid cycle Photosynthesis NADH FADH2 O2 H20 Respiratory chain NH3 CO2 ATP

23. Glycolysis • Series of biochemical reactions that converts 1 glucose molecule to 2 molecules of pyruvate with release of usable E in form of 2 ATP molecules. • An anaerobic process in the cytoplasm • On its own an inefficient metabolic pathway for E generation. • Pyruvate can be further fermented to lactate or ethanol.

24. Glycolysis overview I II III

25. Major stages in glycolysis • I. Conversion of glucose to fructose-1,6-bisphosphate (2 ATP molecules used). • II. Generation of two, inter-convertible 3-carbon molecules, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). • III. Conversion of DHAP and GAP to pyruvate (each 3-carbon molecule oxidised to produce 2 ATP molecules and 1 NADH). Net gain of 2 ATP and 2 NADH molecules.

26. Glucokinase G-6-P Hexokinase G-6-P inhibits Glycogen PPP’way ATP, low pH, citrate, low glucose inhibit. F-2,6BP, AMP, high glucose stimulate Phosphofructokinase Insulin & glucagon regulate Pyruvate kinase Pyruvate Dehydrogenase Glycolysis (in cytosol) Glucose G-6-P F-6-P F-1,6-BP DHAP GAP 1,3-bisphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate PEP Pyruvate Mitochondrion Acetyl-CoA

27. CH2OH O O 2-O3POH2C CH2OPO32- OH H HO OH OH HO HO OH OH H Glucose Fructose 1,6-bisphosphate Glycolysis (stage I) • Hexokinase phosphorylates glucose (G) to G-6P (1 ATP molecule used). • Phosphoglucose isomerase isomerises G-6P to fructose (F)-6P (6 to 5 member ring conversion). • Phosphofructokinase (PFK) phosphorylates F-6P to fructose 1,6-bisphosphate (F-1,6BP) (1 further ATP molecule used).

28. CH2OPO32- CH2OPO32- O C O C + H HO C H HO C H OH Aldolase C H H C OH H O CH2OPO32- C H C OH Fructose 1,6-bisphosphate Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate CH2OPO32- Glycolysis (stage II) • F-1,6BP is split into isomers, DHAP and GAP, by aldolase (highly reversible). • DHAP converted reversibly to GAP by triose phosphate isomerase, hence 1 glucose gives 2 GAP molecules.

29. Phosphoryl transfer potential

30. Glycolysis (stage III) OPO32- O C + NADH + H+ H C OH CH2OPO32- 1,3 Bisphosphglycerate H O C + NAD + pi H C OH CH2OPO32- Glyceraldehyde 3-phosphate - +ATP OPO32- O O O Phosphoglycerate kinase C C + ADP H C OH H C OH CH2OPO32- CH2OPO32- 1,3 Bisphosphglycerate 3-Phosphglycerate • GAP dehydrogenase (GAPDH) catalyses oxidation of GAP to 1,3-bisphoshoglycerate (1,3-BPG), with production of NADH and H+. • 1,3-BPG has high phosphoryl transfer potential. Phosphoglycerate kinase transfers phosphate group to ADP to form 1 ATP molecule. Glyceraldehyde 3-phosphate dehydrogenase

31. - - O H2O H H H H 3-Phosphglycerate Phosphoglycerate mutase Enolase Phosphenolpyruvate Pyruvate 2-Phosphglycerate O O O O C C ADP + H+ O ATP - - H H C C OPO32- OH O OPO32- O O C OH C OPO32- Pyruvate kinase CH3 H H Glycolysis (stage III) • 3-PG is converted to 2-PG by phospho-glycerate mutase. • 2-PG is dehydrated to phosphoenol-pyruvate (PEP) by enolase. • High phosphoryl transfer potential of PEP catalysed to pyruvate by pyruvate kinase (PK) with formation of 1 ATP molecule.

32. Glycolysis • Net energy yield from glucose to pyruvate: • Glucose + 2 Pi + 2 ADP + 2 NAD+ •  2 pyruvate + 2 ATP + 2 NADH + 2H+ + 2H2O • Redox balance and outcome of pyruvate • Reduction of pyruvate and acetaldehdye by NADH to lactate (LDH) and ethanol (ADH) respectively. • Pyruvate transferred to mitochondria, decarboxylated and oxidised to form acetyl CoA. • Pyruvate + NAD+ +CoA  acetyl CoA + CO2 and NADH pyruvate dehydrogenase complex Krebs cycle (aerobic) N.B. Redox balance: NADH cannot be kept at the reduced state for long, needs to be oxidised i.e. release its electrons.

33. Cori cycle • In skeletal muscle undergoing intense contraction and in red blood cell (no mitochondria), pyruvate and NADH accumulate. NAD+ is regenerated by reduction of pyruvate to lactate (LDH). • Lactate diffuses into circulation to liver, where it is oxidised back to pyruvate and subsequently converted into glucose to be fed back to muscle. NADH NAD+

34. Fructose & galactose enter glycolysis p’way at intermediate stages • Fructose is converted by fructokinase to fructose1-phosphate (F-1P). F-1P split into DHAP and GAP by another aldolase, F-1P aldolase. • Alternatively, fructose converted (with less affinity than glucose) by hexokinase to F-6P. • Galactose is converted to glucose-6P in 4 steps (galactokinase, galactose 1-phosphate uridyl transferase, epimerase, phosphoglucomutase). • Galactose + ATP  glucose 6-phosphate + ADP + H+ Sucrose (common table sugar) = fructose and glucose Lactose = galactose and glucose

35. Milk intolerance • Insufficient lactase in the gut to cope with milk intake. • Lactase breaks down lactose to glucose and galactose. • Post-weaning lactase levels down to 5 to 10% of birth. • Microbial breakdown of lactose to lactic acid, methane and hydrogen. Undigested lactose and lactic acid osmotically draw fluid into gut lumen, hence diarrhoea.

36. Regulatory checkpoints of glycolysis • The irreversible rxns of: • hexokinase (G  G-6P) and • phosphofructokinase (PFK) (F-6P  F-1,6BP), • pyruvate kinase (PK) (PEP  pyruvate) are regulatory checkpoints. • The corresponding genes are regulated at the level of transcription and translation. • Their enzymatic activities are regulated by allosteric effects or by covalent modifications. Allosteric site is binding site on enzyme other than the active site. Binding of regulatory molecule to allosteric site changes shape of molecule enabling or preventing binding of substrate. Covalent bonds are formed by sharing of valence electrons

37. Regulation of PFK activity • PFK is a tetramer, each subunit with a catalytic and an allosteric site. • Inhibitors of PFK: • Allosteric ATP binding to PFK reduces affinity of PFK to F-6P. • Rise in [H+] or fall in pH inhibits PFK (as in lactic acid build-up). • Citrate, an early intermediate of the citric acid cycle, enhances the inhibitory effect of ATP. • Low glucose level. • Activators of PFK: • AMP reverses the allosteric inhibition of ATP. • High glucose level. • Allosteric F-2,6BP binding stimulates PFK (Note: not F-1,6BP). hyperbolic sigmoidal N.B. Glycolysis degrades glucose to generate ATP & provides building blocks for synthetic rxns e.g. forming long chain fatty acids. High level citrate means biosynthetic precursors are abundant

38. Allosteric activator: F-2,6BP • Glucose high: PFK2 (dual kinase and phosphatase) converts F-6P to F-2,6BP, which in turn stimulates PFK (note PFK2  PFK). • Glucose low:  glucagon   protein kinase A  phosphorylation of PFK2  activation of its phosphatase domain  reduced F-2,6BP  reduced PFK activity (in liver). (Glucagon promotes glycogenolysis and gluconeogenesis)

39. Hexokinase • Hexokinase (G  G-6P) is inhibited by G-6P. • Inhibition of PFK (F-6P  F-1,6BP) will eventually lead to build-up of G-6P. Hence inhibition of PFK will also inhibit hexokinase. • Role of isozyme glucokinase (Gk, lower affinity for glucose and not inhibited by G-6P): • When glucose high, Gk provides G-6P for glycogen synthesis (G-6P can also be oxidised by pentose phosphate pathway to generate NADPH).

40. Pyruvate kinase • PK (PEP  pyruvate + ATP), a tetramer, encoded by different genes to give rise to L-type (liver isoform) and M-type (muscle and brain isoform). • Activators of PK: • F-1,6BP (product of PFK) activates both PK isoforms. • Insulin signalling leads to dephosphorylation (activation) of PK (see later). • Inhibitors of PK: • ATP allosteric inhibition of both PK isoforms. • Alanine (made from pyruvate) allosteric inhibition • abundance of building blocks. • Glucagon signalling leads to phosphorylation (inactivation) of L isoform of PK.

41. Pi Phosphorylated pyruvate kinase (less active) H20 ADP ATP Pi Pyruvate + ATP Phosphoenolpyruvate + ADP + H+ Covalent modifications of pyruvate kinase (liver) under high and low glucose status. Insulin Dephosphorylated Pyruvate kinase (active) Glucagon

42. Pentose phosphate pathway Cytosol • Primarily anabolic. Uitilises 6 carbons of glucose 5 carbon sugars and reducing equivalents • However, does oxidise glucose & under some conditions can completely oxidise glucose to CO2 and H2O • PPP pathway branches from glycolysis at level of G-6-P

43. Primary functions PPP • To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis. • To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. Reductive biosynthesis

44. NADPH for reductive biosynthesis. • In nucleated cells w/ active lipid biosynthesis e.g. lactating mammary glands, adrenal cortex and liver • NADPH is used in redox rxns required for biosynthesis of fatty acids, cholesterol, steroid hormones and bile salts. • In liver • NADPH used for hydroxylation reactions involved in detoxification and excretion of drugs • In RBCs • NADPH used in reduction of glutathione

45. Secondary function • Metabolise dietary pentose sugars (5 carbon atoms) derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates

46. PPPathway also described as a shunt • When pentoses not needed for biosynthetic rxns, pentose phosphate intermediates are cycled back into mainstream of glycolysis by conversion in F-6P and glyceraldehyde-3-phosphate Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD+/NADH cofactor pair. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione. The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source, therefore, any rapidly proliferating cell needs large quantities of NADPH.