Chemoorganoheterotroph Metabolism Overview:

1. Chemoorganoheterotroph Metabolism Overview: Reduction; e- gain from donor Oxidation; e- loss to acceptor

2. Metabolic Pathways • Although we can recognize a substrate and product of individual enzymatic reactions; metabolic functions are often performed by several enzymatic reactions in a “pathway”. • Pathways can be linear, branched, cyclic or even spiral. • Pathway activity is controlled in three ways: • Metabolites and enzymes may be localized in different parts of the cell; called metabolic channeling. • The total amount of enzymes in a pathway can vary (gene expression). • Pathway activity is controlled by critical regulated enzymes. These “pacemaker enzymes” are often the rate-limiting step in the pathway.

3. Metabolic Pathways • Feedback Inhibition: (“end-product inhibition”) • rate limiting enzyme is first in pathway and allosteric. • end-product is a negative effector (inhibitor) of first enzyme

4. Metabolic Pathways • Feed Forward Activation: • rate limiting enzyme of a branch point is allosteric. • earlier-substrate is a positive effector (activator) of forward reaction enzyme. • NOTE: the example also illustrates feedback inhibition. +

5. Metabolic Pathways • Amphibolic Pathways • Catabolic direction • Anabolic direction • Separate regulatory enzymes each way; function as “check valves” for flow control. • Other pathway enzymes are reversible; ΔGo’≈0; their equilibrium shifts based on concentration of reactants & products. • Gycolysis is a good example.

6. Catabolism: • Hydrolysis of complex (polymeric) organics • Breakdown of Glucose (6C) to Pyruvate (3C) • Glycolytic Pathway (Embden-Meyerhof) • Pentose Phosphate Pathway (PPP) • Entner-Doudoroff Pathway (E-DP) • Fate of Pyruvate • Fermentation • Krebs Cycle (Tricarboxylic Acid, i.e. Citric Acid) • Electron Transport Chain & Oxidative Phosphorylation • Aerobic Respiration • Anaerobic Respiration

7. Hydrolysis of Complex Organics • Role of exo-enzymes and enzymes of cytoplasm or lysosome. • Glucosidase (polysaccharides → simple sugars) • Proteases (proteins → amino acids) • Nucleases (nucleic acids → nucleotides) • Lipases (lipids → glycerol & fatty acids) • Hydrolysis reactions yield little energy & mostly lost as heat. • Simple sugars (e.g. glucose) enter catabolic reactions early at Glycolysis, PPP, E-DP, or used to biosynthesize other polysaccharides. • Others may be degraded to some intermediate of a major catabolic pathways (e.g. fatty acids to acetyl-CoA) or used directly in anabolic reactions (e.g. some amino acids).

8. Catabolism of Macromolecules • Carbohydrates: • Some monosaccharides may require interconversion to glucose or other sugar intermediate. • C5 & C4 sugars to PPP. • Proteins: • Amino acid deamination and transamination. • Mostly enter pathways as carboxylic acid intermediates. • Nucleic Acids • Ribose sugar to PPP • Purines and Pyrimidines to component amino acids. • Lipids • Glycerol and fatty acids • Fatty acids to acetyl-CoA via β-oxidation.

9. Catabolism of Glucose Glycolosis: Both R & F; ends in pyruvate; ATP via substrate level phosphorylation (SLP). Respiration: pyruvate oxidized by Krebs Cycle (ATP by SLP); Electron Transport Chain & Chemiosmosis (ATP via oxidative phosphorylation); terminal electron acceptor (O2 or other like NO3-). Fermentation: Lack of respiration; pyruvate is reduced to another organic; little ATP yield by SLP. Fermentation only 2-4 ATP Aerobic Respiration 38 ATP

10. Glucose → Pyruvate via Glycolysis Six Carbon (Investment) Stage: Hexose phosphorylation and cleavage to two Glyceraldehyde-3-Phosphates. Five enzymatic reactions in this stage; three are reversible. Enzyme #1: Hexokinase (- G6P) & Enzyme #3: Phosphofructokinase (+ AMP; - ATP; - Citrate) are regulated enzymes. Requires an investment of 2 ATP to “prime” the reaction.

11. Glucose → Pyruvate via Glycolysis Three Carbon (Yield) Stage: Two glyceraldehyde-3-phosphates can pass per glucose. Five more enzymatic reactions for a total of ten in Glycolysis. Four at this stage are reversible. Enzyme #10: Pyruvate Kinase (+ F1,6BP; - ATP) is regulated. As the carbon becomes oxidized the phosphate bonds elevate in their energy potential. Two steps involve SLP for ATP. Total of 4 ATP yield here, minus 2 ATP invested earlier = 2 ATP for all of Glycolysis, plus 2 NADH.

12. Glucose → Pyruvate via E-DP Two stage linear pathway like Glycolysis; First stage unique; second stage identical. KDPG the unique intermediate. Yields 1 ATP, 1 NADH, 1NADPH. Note one pyruvate is generated at each stage of the pathway. Found in some Gram negative bacteria instead of Glycolysis (Rhizobium, Agrobacterium, Azotobacter, Pseudomonas) (KDPG)

13. Glucose → Pyruvate via PPP Found in most bacteria. Can produce pentoses (5C) from hexoses (6C) via oxidative decarboxylation, which forms NADPH. Source of ribose for nucleosides. Other unique sugars are produced (4C, 7C); source of erythrose for aromatic amino acids (Phe, Tyr, Trp) G3P enters Gycolysis to produce ATP and pyruvate. (GP) (GP)

14. Fermentation: Lack of any respiration accumulates NADH. Without NAD+, Glycolysis or E-DP won’t proceed, i.e. no source of ATP! Fermentation pathways couple NADH oxidation and pyruvate reduction, or reduction of another endogenous organic. Permits some ATP production; slow growth. Many species specific types.

15. Krebs (TCA) Cycle Respiration allows pyruvate to be completely oxidize to CO2. First oxidative decarboxylation converts pyruvate to Acetyl-CoA by the multi-enzyme Pyruvate Dehydrogenase Complex – highly regulated “pacemaker”. Acetyl-CoA (2C) forms citric acid (6C) by condensation with oxaloacetate (4C). During the next 7 cyclic steps: Two additional decarboxylations. 1 ATP by SLP. 3 NADH & 1 FADH2 produced. Back to 1 oxaloacetate Two cycles are required per glucose.

16. Β-Oxidation of Fatty Acids The spiral pathway of fatty acid synthesis is nearly the reverse of this.