1. 1 Central metabolic pathways: Pathways that provide precursor metabolites to all other pathways Carbohydrates metabolic pathways and carboxylic acids  Embden – Meyerhof – Parnas Pathway= EMP = glycolysis  Pentose phosphate pathway(PPP)  Entner Doudoroff pathway(ED) Similarities of the three pathways: Convert glucose to phosphoglycer- aldehyde Phosphoglyceraldehyde is converted to pyruvate through the same reactions Carbohydrates pathways and Krebs Cycle Central metabolic pathways: Three substrate level phosphorylations Six oxidation reactions → NADH and FADH2

2. Pyruvate → depends on cell Respiration → oxidized to acetyl- CoA → CO 2 Fermentation → alcohol, organic acids, solvents. The oxidation of NADH and FADH 2 Respiration → oxidized via electron transport → formation of ∆ P Fermentation → oxidized in cytosol by an organic acceptor → no production of ATP Glucose6-P-Gluconate ATP G6P NADPH ATP PPPED ATP CO 2 NADPH Pentoses Pi H 2 O F6P ATP FBP Gluconate Fumarate Succinate ATP, NADH ATP CO 2 NADH citrate Cis- aconitate Isocitrate FADH 2 ATP succinyl CoA CO 2 NADH 2 α-ketoglutarate NADPH oxalosuccinate CO 2 PGLAD PEP Pyruvate Acetyl-CoA Oxaloacetate Malate NADH Citric Acid Cycle

3. 3 Embden – Meyerhof – Parnas Pathway Catalyzes the splitting of the glucose molecule (C 6 ) into two phosphoglyceraldehyde (C 3 ) molecules Catalyzes the oxidation of phosphoglyceraldehyde (C 3 ) to pyruvate Figure 1. Glycolysis

4. Glycolysis as an anabolic pathway The glycolytic pathway serves not only to oxidize carbohydrate to pyruvate and to phosphorylate ADP, but also provides precursor metabolites for many other pathways. G6P → polysaccharides, aromatic amino acids, pentosa phosphates F6P → amino sugars (muramic acid and glucosamine) DHAP → phospholipids PGA → serine, glycine, cystein PEP → aromatic amino acids, muramic acid 4 Glycolytic pathway can be reversed from PEP only to F 1,6 bP (not at all to pyruvate). This is due to the pyruvate kinase and phosphofructokinase reactions are physiologically irreversible → high free energy in the phosphoryl donors Regulation of glycolysis pathway: Two key enzymes : 1. phosphofructokinase 2. fructose 1,6 bisphosphate phosphatase

5.  phosphofructokinase activity will be stimulated when the ADP levels are high (ATP levels are low) → glycolysis is stimulated  phosphofructokinase activity is inhibited by PEP (end - product inhibition). When glycolysis is stimulated by ADP, the reversal of glycolysis is slowed by high levels of AMP → AMP inhibits the fructose 1,6 bisphosphate reaction Fructose 1,6 bisphosphatase regulates pyruvate kinase 5 Pentose Phosphate Pathway In this pathway: Producing pentose phosphate → precursors to the ribose and deoxyribose in the nucleic acid Providing erythrose phosphate → precursors to the aromatic amino acids

6. Reactions of Pentose phosphate pathway:  Oxidation-decarboxylation reactions products : CO 2, 2 NADPH, 5 carbon sugar (ribulose –5– phosphate).  Isomerization reactions → precursors to the stage 3 some of the ribulose-5-phosphate is isomerized to ribose-5-phosphate and to xylulose-5-phosphate Two type of reactions: Transfer of two-carbon fragment from ketose to an aldose → transketolase Transfer of three-carbon fragment from ketose to an aldose → transaldolase The rule : the donor is always a ketose and the acceptor is always an aldose  Sugar rearrangement reactions → glyceraldehyde phosphate. 6

7. Thiobacillus novellus and Brucella abortus use only an oxidative pentose phosphate pathway to grow on glucose Stage 2 and 3 of the pentose phosphate pathway are reversiblesynthesize pentose phosphates from phosphoglyceraldehyde The pentose phosphate pathway and glycolysis interconnect at phosphoglyceraldehyde and F6P organisms growing on pentoses can make hexose phosphates Figure 3. Pentose phosphate pathway 7

8. Entner--Doudoroff Pathway  Only found in prokaryotes. widespread, particularly among gram-negative bacteria, aerobic  Yield of the pathway: 1 mol glucose2 pyruvate, 1 ATP, 1 NADH and 1 NADPH.  Difference to pentose phosphate = some of 6P–gluconates are dehydrated to 2 keto-3-deoxy-6- P-gluconate (KDPG) instead of to ribulose-5-phosphate. Figure 4. Entner-Doudoroff pathway 8

9. The Citric acid cycle: This cycle is present in most heterotrophic bacteria growing aerobically, but organisms that grow on C1 compounds (methan, methanol and so on) carry out a reductive pathway They are 4 oxidations per acetyl- CoA producing2 NADH, 1 CITRIC ACID CYCLE NADPH and 1 FADH2, and 1 substrate-level phosphorylation producing ATP. CoA 9

10. The Citric acid cycle is feedback inhibited by several end product:  In Gram – bacteria: citrate synthase is allosterically inhibited by NADH.  In facultativ anaerob by α- ketoglutarat.  In Gram + the citrate synthase is inhibited by ATP The Citric acid cycle provide precursors to 10 of the 20 amino acid found in protein: Succinyl-CoA: L- lysine and L- methionin, tetrapyroles (prosthetic group in several protein, Cyt & Chlorophylls) 10 Oxaloacetate : aspartate, which itself is the precursor to five other amino acid. Fumarate: aspartate  -ketoglutarat :glutamate, which itself is the precursor to three other amino acid. Modification of the citric acid cycle into a reductive cycle during fermentation Growth. The solution is to convert the citric acid cycle from an oxidative into a reductive pathway.

11. 11 Pyruvate Acetyl-CoA Oxaloacetate Citrate malate Fumarate succinate Succinyl~CoA [ cis-akonitate] isocitrate oxalosuccinate α -ketoglutarat Glucose-6-P The Glyoxylate Cycle Phosphoenolpyruvate The Glyoxylate cycle is required by aerobic bacteria to grow on fatty acids and acetate (Plants and protozoa also have this cycle) What regulates the fate of the isocitrate? In E. coli: the isocitrate hydrogenase activity is partially inactivated by phosphorylation when cell are grown on acetate.

12. Acetate also induces the enzymes of Glyoxylate cycle. Isocitrate lyase requires a high intracellular concentration of isocitrate Gluconeogenesis Growing microorganisms on poor carbon source, suchas L-malate, succinate,acetateorglycerol requires the ability to synthesize hexoses needed for the production of cellwallmucopeptides,storage glycogenandothercompounds derivedfromhexose,suchas pentoses,fornucleicacid biosynthesis 12

13. 13 Hexose synthesis involves a reversal of carbon flow from pyruvat (gluconeogenesis). It is not allow a carbon flow from pyruvat to hexoses directly because: 1. Pyruvat kinase is not reversible because the free-energy requirement is to great. PEP is formed by PEP-carboxykinase (Mg 2+ dependent, with GTP as phosphate donor) 2. Reaction with phosphofructokinase is irreversible. Fructose-1,6- bisphophatase dephosphorylates FBP to yield F-6-P and Pi. 3. Glucose – 6- phosphatase removes Pi from G-6-P to yield glucose. 2 pyruvate + 4ATP + 2GTP + 2NADH + 2H + + 4H 2 O →  Glucose + 2NAD + + 4ADP + 2GDP + 6Pi

14. 14 Gluconeogenesis enzymes 1. Pyruvate carboxylase: pyruvat → OAA 2. PEP carboxykinase: OAA + GTP → pyruvat 3. Fructose-1,6-bisphosphatase 4. Glucose-6-phosphatase 5. ATP-glucose pyrophosphorylase: G- 1-P +ATP →  ADP-glucose 6. Glycogen Synthase: α -1,4-glycan + ADP-glucose →  glycogen / starch

15. 15 Regulation: carboxykinase, which is regulated by catabolite repression (inhibited when glucose or other carbohydrate carbon source are available). PEP carboxykinase is induced at the stationary phase of growth and requires cAMP and regulatory signal. Regulation: 1. Allosteric Control of enzymes activity in Catabolic Pathways The major regulatory step is PEP Alosteric regulation in Emden-Meyerhof- Parnas, Gluconeogenesis and TCA is predominantly effected by intermediates. This is due, in part,to the interrelation of the Emden-Meyerhof-Parnas, pentose phosphate and TCA pathways at the level of intermediates of Emden- Meyerhof-Parnas

16. 16  The effector intermediates may either be inhibitors, activators, or both (affecting different enzymes)  accumulation of Fru 1,6 biP serves to activate both ADP- glucose pyrophosphorylase and pyruvate kinase  accumulation of PEP serves to inhibit phosphofructokinase but activates pyruvate dehydrogenase

17. 17 2. Posttranslational Covalent Modification of proteins  Covalent modification of prokaryotic proteins involves phosphorylation at various residues, uridylylation, adenylylation, methylation, fatty acylation and proteolysis  Example: Isositrat Dehydrogenase For growth on acetate or fatty acids, E.coli require the functioning of the glyoxylate bypass. During growing on acetate, isocitrate dehidrogenase is inhibited by approximately 75% compared to during growth on most other carbon.

18. 18 This inhibition is caused by the phosphorylation of a single serine residue of isocitrate dehydrogenase, which completely inactivates the enzyme. Phosphorylation of isocitrate dehydrogenase is carried out by an ATP-dependent IDH kinase/ phosphatase, which also catalyzes dephosphorylation of phospho- isocitrate dehydrogenase.