1. Chapter 6
Cell Metabolism

2. 1
Metabolism 2
Regulation of metabolism
Errors in metabolism 3
Biotechnology applications 4

3. Metabolic Pathways Metabolic Pathways
Break down and manufacture molecules in a sequential set of reactions
Enzyme reaction: generate products from substrates
Branch and converge, and form networks
Similar metabolic pathways from bacteria to human

4. Catabolism and Anabolism
Breaking down, Energy-yielding metabolism
Energy release from bond breakage
Burning of gasoline : Conversion to molecules with lower energy in bond
Burning of fat in human body : Enzymatic generation of molecules with lower energy in bond
Anabolism
Synthesis, Energy-requiring metabolism

5. Catabolism and Anabolism

6. Catabolism of Food in Human Body
Digestive system
Breaking down carbohydrates, lipids, and proteins into building blocks
Sugars
Used as E source immediately
Stored as glycogen for short term storage (liver, muscle) : 1 to 2 days
Fatty acids
Stored as fats for long term storage (fat cells): 4-6 weeks
Amino acids
Used for protein synthesis, generation of other amino acids
E source

7. Catabolism of Glucose Glycolysis Aerobic conditions
From bacteria to animals
Glucose (C6) to two pyruvic acid (C3)
No O2 is required
Aerobic conditions
Conversion of pyruvic acid to CO2 and acetyl coenzyme A (acetyl-CoA)
TCA (Krebs cycle)
Acetyl CoA  CO2 + H2O + NADH (temporary storage molecule)
Anaerobic conditions
Fermentation
Generation of ethanol in yeast
Lactic acid synthesis in muscle

8. Catabolism of Glucose

9. NADH and NADPH NAD(P) Nicotinamide adenine dinucleotide (phosphate)
Accept hydride (:H-) released from oxidation (dehydrogenation) of substrate : either A side or B side, not both sides
NAD(P)+ + 2H+ + 2e-  NAD(P)H + H+
NAD(P)+ : + indicates oxidized form, not the net charge of NAD(P) which is -
Benzenoid ring
Quinonoid ring

10. Flavin Nucleotide
370 and 440 nm absorption

11. Electron Transport Pathway
Generation of ATP as energy storage molecule
ATP : high E Phosphodiester bond
Reduction of O2 to H2O
NADH + H+ + 3ADP + 3Pi + ½ O2  NAD+ + 3ATP + H2O
FADH ADP + 2Pi + ½ O2  FAD + 2ATP + H2O
c.f. cyanide: blocking electron transport pathway

12. Chemiosmotic Model Proton motive force derives ATP synthesis
Passive proton flow into matrix through proton pore associated with ATP synthase

13. Energy transducing membranes
Bioenergetics4 by D. Nicholls

14. Net Reaction Glycolysis Fermentaion TCA cycle
Glucose + 2ADP + 2Pi + 2NAD+  2 Pyruvate + 2ATP + 2NADH + 2H H2O
Fermentaion
2 Pyruvate + 2NADH + 2H+  2 Lactase + 2NAD+
TCA cycle
Pyruvate + 4 NAD+ + FAD + ADP + pi + 2H2O  4 NADH + 4 H+ + FADH2 + ATP + 3 CO2
Glycolysis + fermentation : 2ATP
Glucose + 2ADP + 2Pi  2 Lactase + 2ATP
Glycolysis + TCA cycle : ~ 38 ATP
Glucose + 4ADP + 4Pi + 10NAD+ + 2 FAD + 4H2O  4 ATP + 10 NADH (30 ATP) + 10H+ + 2 FADH2 (4 ATP) + 6 CO2

15. ATP Synthase : F1
a3b3gde
Knoblike structure with alternating a and b arrangement
g subunit
Central shaft
Association with one of the three b subunits (b-empty)
Induction of different conformation of b subunits
Difference in ADP/ATP binding sites of b subunits
b-ATP, b-ADP, b-empty

16. ATP Synthase : Fo ab2c10-12 C subunit
Small (Mr 8,000) hydrophobic two transmembrane helices
Tow concentric circles
Inner circle : N terminal helices
Outer circle (55 Å in diameter) : C terminal helices

17. Rotational Catalysis Mechanism
Binding change of b subunit (Paul Boyer)
Stationary membrane attachment a3b3 spheroid through b2 and d subunits
Proton passage through Fo
Rotation of the cylinder of c subunits and g subunit
Every rotation of 120o: g contacts with a different b subunit
b subunit contacting g : b-empty
Neighboring b : b-ADP or b- ATP
Cycle of b-ADP  b- ATP  b-empty
ATP synthesis
Apposite rotation of g
ATP hydrolysis

18. Detection of the Rotation of g Subunit H. Noji et al., 1997, Nature
Purification of a,b,g subunits of thermophilic bacterium in E. coli
N terminus of b subunit tagged with His
attached to glass coated with Ni-NTA
Biotinylation of g subunit by introducing Cys residue
Biotinylation and fluorescent label of actin
Assembly of g subunit and actin by streptavidin
4 binding sites for biotin
Detection g subunit rotation under a fluorescence microscope in the presence of ATP

19. Energy and Torque Considerations
ATPase
Hydrolysis of 300 molecules of ATP/s
100 revolutions/s
Torque; >40 pN nm
Efficiency of the rotor : ~ 90%
Energy required for 120o rotation: 8 X J
Free energy generated by ATP hydrolysis: 9 X 10-20J

20. Stoichiometries of O2 Consumption and ATP Synthesis
xADP + xPi +1/2O2 + H+ + NADH  xATP + H2O + NAD+
X : P/O ratio, P/2e- ratio
Before the chemiosmotic model
P/O is integer
3 for NADH and 2 for succinate
After the chemiosmotic model
No requirement for P/O to be integer
2.5 for NADH and 1.5 for succinate
Proton efflux : 10 for NADH, 6 for succinate
4 protons for 1 ATP synthesis

21. ATP yield of complete oxidation of Glc

22. Catabolism of Other Nutrient

23. Anabolism Requirement Energy: ATP
Chemical building blocks (intermediates of glucose breakdown)

24. Metabolism 1
Regulation of metabolism 2
Errors in metabolism 3
Biotechnology applications 4

25. Regulation of Metabolism
Feedback Inhibition
Inhibition of enzyme activity by end product
e.g. amino acid synthesis

26. Positive regulator vs. negative regulator

27. Regulation of Metabolism by Gene Expression
Trp synthesis in E.coli( Trp operon)
Turn off transcription of Trp genes in the presence of Trp
Hormonal regulation in higher eukaryotes

28. Lac operon

29. 1
Metabolism
Regulation of metabolism 2 3
Errors in metabolism 3
Biotechnology applications 4

30. Errors in Metabolism Enzyme defects and amino acid metabolism
Phenylketonuria (PKU)
Phenylalanine hydroxylase (PAH) defect
No conversion of phenylalanine to tyrosine
Production of phenylketones
Excretion of phenylalanine and phenylketones in the urine
Phenylalanine inhibits normal development of nervous system
Treatment with controlled diet
Alkaptonuria
Defect in enzyme converting homogentisatate (HG) to maleylacetoacetate (MAA)
Oxidation of HG leads to black color  black urine
No serious effect
Albinism
Lacking enzyme converting tyrosine to melanin
Cretinism
Lacking enzyme converting tyrosine to thyroid hormone
Defect in growth and maturation of the skeletal and nervous systems

31. Diseases Related to the Defects in Phe Metabolism

32. 1
Metabolism
Regulation of metabolism 2
Errors in metabolism 3
Biotechnology applications 4 4

33. Biotechnology Applications
Treatment of Metabolic Disorders
Gaucher’s disease
Problems
Defect in enzyme breaking down lipid glucocerebroside in RBC and WBC
Macrophage cannot break down glucocerebroside in engulfed blood cells
Accumulation of enlarged macrophage (Gaucher cell) in spleen, liver, and bone marrow, sometimes in nervous system
Treatments
Enzyme replacement of recombinant enzymes
Gene therapy

34. Using Microbial Metabolism
Using enzymes for manufacturing (Biocatalyst)
Biotransformation
Whole cell reaction
e.g. production of fermented foods (wine, beer, cheese)
Reaction with isolated enzymes
Bioremediation
Using microbes to degrade pollutants
e.g.oil-eating microbes

35. Generation of Useful Products from Microbial Metabolism

36. Enzymes in manufacturing
Invertase: soft-centered chocolate
Cellulase: stone-washed jeans
Amylase: reduced-calorie beer
Converting starch to sugars that yeast can use during brewing process
Lipase, proteinases : laundry detergents