Chapter 6 Cell Metabolism

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

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

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

Catabolism and Anabolism

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

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

Catabolism of Glucose

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

Flavin Nucleotide 370 and 440 nm absorption

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 FADH2 + 2 ADP + 2Pi + ½ O2  FAD + 2ATP + H2O c.f. cyanide: blocking electron transport pathway

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

Energy transducing membranes Bioenergetics4 by D. Nicholls

Net Reaction Glycolysis Fermentaion TCA cycle Glucose + 2ADP + 2Pi + 2NAD+  2 Pyruvate + 2ATP + 2NADH + 2H+ + 2 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

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

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

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

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

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 10-20 J Free energy generated by ATP hydrolysis: 9 X 10-20J

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

ATP yield of complete oxidation of Glc

Catabolism of Other Nutrient

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

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

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

Positive regulator vs. negative regulator

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

Lac operon

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

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

Diseases Related to the Defects in Phe Metabolism

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

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

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

Generation of Useful Products from Microbial Metabolism

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