Introduction to Metabolism

Metabolism (The Acquisition and Utilization of Free Energy) Catabolism: exergonic oxidation Anabolism: endergonic processes

Endergonic Processes Mechanical Work Active Transport Biosynthesis

Anabolism and Catabolism exergonic endergonic

Roles of ATP and NADP + in Metabolism

ATP Kinetic Stability of Phosphoanhydride Bonds

ATP Adenosine Ribose Triphosphate

Hydrolysis of ATP

Phosphate Compounds

Roles of ATP (Coupled Reactions) ∆G o’ (kJ/mol) Fructose-6-P + P i ——> Fructose-1,6-bisP + H 2 O ATP + H 2 O ——> ADP + P i Fructose-6-P + ATP ——> Fructose-1,6-bisP + ADP -17.2

Roles of ATP Early stages of nutrient breakdown Glucose + ATP ——> Glucose-6-P + ADP Interconverson of nucleoside triphosphtes NDP + ATP ——> NTP + ADP Nucleoside Diphosphate Kinase Physiological processes –Muscle contraction –Active transport

Roles of ATP Additional phosphoanhydride cleavages in highly endergonic reactons (NMP) n + NTP ——> (NMP) n+1 + PP i PP i + H 2 O ——> 2 P i Pyrophosphatase

Sources of ATP Phototrophs: photosynthesis Chemotrophs: oxidation of organic compounds (e.g. carbohydrates, lipids, and proteins)

Formation of ATP Adenylate Kinase reaction 2 ADP ——> AMP + ATP Substrate-level phosphorylation X–P + ADP ——> X–H + ATP Oxidative phosphorylation Photophosphorylation

Substrate-Level Phosphorylation

Oxidative Phosphorylation

Photophosphorylation

Source of NAD(P)+, and other cofactors

NADP + Nicotinamide Adenine Dinucleotide (Phosphate)

Figure 14-1 Niacin

Figure Reduction of NAD + or NADP + to NADH or NADPH

Metabolic Pathways A ——> B ——> C ——> D ——> E Metabolites Enzymes

Metabolic Map

Figure 14-3 Overview of Catabolism

Properties of Metabolic Pathways Separate Anabolic and Catabolic Pathways Steady-State Irreversible (overall): reversibility of individual steps First Committed (Exergonic) Step: others close to equilibrium Compartmentation (organelles & tissues): isoenzymes and transport Regulation (usually first committed step): often rate-limiting

Potential Futile Cycles (Regulation)

Steady State

Thermodynamics of individual steps A  B  G o’ = -RTlnK eq Not standard conditions or at equilibrium:  G =  G o’ +RTln([B]/[A]) Three Physiological Conditions:  G o’ <<<<<<0 :  G always negative Example: ATP hydrolysis  G o’ >0 : near equilibrium, reversible, direction depends on actual [B]/[A] Example: Most reactions  G o’ >>>>>>0 :  G always positive, must be coupled Example: Phosphorylation of Glucose

 G o’ >0

Regulation of Metabolic Pathways Specific Controls General Controls

Specific Controls Control of Enzyme Amount –Constitutive Enzymes –Inducible Enzymes –Repressible Enzymes Control of Enzyme Activity –Regulatory Enzymes –Effectors (Ligands)

General Controls (Integration of Cellular or Organism Functions) Internal Effectors –Catabolite Repression –Energy Charge –Reduction Potential External Effectors (e.g. hormones) Significance: Efficiency and Flexibility!

Types of Reactions

Group Transfer Reactions

Phosphoryl Group Transfer

Elimination Reactions

Isomerization Reactions (Intramolecular Hydrogen Shifts)

Making C-C Bonds Note: thioester

Breaking C-C Bonds

Oxidation-Reduction Reactions SH 2 + NAD + + H 2 O ——> S + NADH + H 3 O + SH 2 : Reduced Substrate S: Oxidized Product NAD + : Electron Acceptor FAD: Electron Acceptor

Figure Reduction of NAD + to NADH

Figure Flavin Adenine Dinucleotide (FAD)

Figure part 1 Reduction of FAD to FADH 2

Figure part 2 Reduction of FAD to FADH 2

One Electron Oxidation-Reduction Reactions

Half-Reactions Oxidation Involves(e - of H: - ) Loss Reduction Involves(e - of H: - ) Gain

Alcohol Dehydrogenase (Oxidation-Reduction Reaction)

Experimental Approaches to Metabolism

Features of Metabolic Pathways A ——> B ——> C ——> D ——> E (1)Sequences and Energetics (2) Enzymes and Mechanisms (3) Control Mechanisms (Regulation) (4) Compartmentation

Elucidation of Metabolic Pathways A ——> B ——> C ——> D ——> E Metabolic Inhibitors: accumulation of intermediates Biochemical Genetics: mutants Pathway Labeling: isotopes

Metabolic Inhibitors (Accumulation of Intermediates) (e.g. Glycolysis) Fluoride: (2-phosphoglycerate and consequently 3–phosphoglycerate)

Biochemical Genetics (Mutants) Natural Genetic Defects Manipulation of Microorganisms Accumulation of Intermediates Growth Requirements (auxotrophic mutants) A ——> B ——> C ——> D ——> E

Pathway Labeling A* ——> B* ——> C* Stable Isotopes Radioisotopes

Detection of Isotopes Stable Isotopes –Mass Spectrometry –NMR Radioisotopes –Proportional Counting (Geiger Counter) –Liquid Scintillation Counting –Autoradiography

Quantify Differential Expression Condition 1Condition 2 Sample Prep Mix samples and detect Quantify Differences

Control of Expression Transcription: –Microarray Proteomics –2D-SDS-PAGE –Isotope Coded Affinity Tag

ICAT Chemistry IAM Biotin Reactive Group (specific for cysteines) Affinity Tag Isotope code (D or 13 C) LIGHT HEAVY ICAT = Isotope Coded Affinity Tag Same behavior chemically, but different in mass.