BiochemistryChapter 14: Introduction to Metabolism- Metabolism consists of anabolic and catabolic pathways - Catabolism : degradative, Usually energy-yielding- Anabolism : biosynthetic, Usually energy-requiring- Organisms classified by how they use energy1. carbon source-autotrophs use CO2 as carbon source-heterotrophs - use C from organic source such as glucose2. energy source-phototrophs - use light as energy source-chemotrophs - use oxidizable chemicals as energy sourceRole of O2: -ultimate electron acceptor in oxidation of energy sources for large number oforganismsaerobes - ultimate e- acceptor is O2anaerobes - different e- acceptor like sulfate or nitrate- obligate anaerobes are poisoned by oxygen; must live in a reducing environment-flow of energy in biosphere can be seen as equilibrium between photoautotrophs and heterotrophsVitamins and minerals are involved in metabolic reactions: - classified as water soluble or water insoluble- water-soluble usually converted into coenzymesparticipate in metabolic reactions-fat soluble (water insoluble) have varied functions, antioxidant, light absorption, etc.Synthesis and degradation pathways use unique energy shuttles:- Degradation - breakdown of highly reduced molecules to produce ATP and NADPH- biosynthetic - use ATP/NADPH to produce reduced molecules and NADP and ADPOrganization in Pathways:Pathways consist of sequential steps- intermediates and products in metabolism are called metabolites- many pathways converge on two central metabolites: pyruvate, acetyl-CoA- glycolysis and the TCA cycle are central to metabolism oxidative pathways: go from highly reduced molecules to CO2Metabolic pathways are compartmentalizedcellular compartments: cytosol - glycolysis, pentose phosphate pathway, fatty acid biosynthesis,some of gluconeogenesismitochondria - TCA cycle, oxidative phosphorylation, fatty acid oxidation,amino acid breakdownsmooth ER - lipid and steroid biosynthesis- expression of metabolic enzymes differ with different tissues- liver is largely responsible for gluconeogenesis- different tissues express different isozymesisozymes are enzymes with the same function but different kinetics orregulationDirection of metabolism dictated by thermodynamics-pathways have multiple steps with multiple enzymes-flow in one direction; have “committed first step” (first rxn in pathway has large negative ΔG)- catabolic and anabolic pathways with same endpoints must use different intermediates for one or more steps in pathways-some reactions are near equilibrium, some are far from equilibrium- can be distinguished by their ΔG°’- rxns with large negative ΔG°’ are at control points in pathwaysenzymes at key steps are regulated by:allosteric control - feedback inhibition, products inhibit earlier rxnscovalent modification - phosphorylation/dephosphorylation can change enzyme kineticssubstrate cycling - different enzymes for forward and reverse pathways(can change reactant/product ratios by changing reaction rates)genetic control - control transcription of metabolic enzymesEnergy is stored/shuttled between pathways via high-energy compoundsHigh-energy compounds are often phosphorylatedATP: high energy compoundhydrolysis of γ phosphate has large negative ΔG3 major thermodynamic contributors to negative ΔG:1. resonance stabilization of ATP is less than that of products2. electrostatic repulsion of negative phosphates3. smaller solvation energy of phosphoanhydrideenergy from high-energy compounds used in coupled rxns; use energy from high-energy compound to give an initially unfavorable reaction a favorable ΔGphosphocreatine: high-energy compoundused to generate ATP during intense activity; acts as ATP buffer-phosphates can easily be swapped btw ATP and other NDPs- ATP can be used to regenerate GTP, CTP, etc. from GDP, CDP and so on- catalyzed by nucleoside diphosphate kinaseATP+NDP->ADP+NTPother kinases generate NDPs from NTPs and NMPs e.g. adenylate kinase AMP+ATP->2ADPAcetyl CoA :high energy compoundthioester bond is high energy; intermediate in multiple pathways-participates in acetyl group transfers-boosts the energy to help rxns proceedOverall metabolic pathways involve redox reactions:in catabolism - move electrons from long energy rich compounds (e.g. fats, sugars) to O2 to make CO2 and H2OThe role of NAD + and FAD: - NAD+ and FAD collect and shuttle electrons released in catabolism to O2- oxidation of reduced carbons leads to reduction of NAD+ molecules to NADH-NAD+ - involved in 2 electron reductions- FAD involved in 1 or 2 electron reductions- reduced NADH and FADH2 are consumed in the TCA cycle to ultimately make ATPFree energy of redox rxns described by Nernst equation- relates redox potentialto ΔGΔE- electrical potential difference; measures likelihood of electrons transferring btw molecules in a redox rxn.An+ + B←⎯⎯⎯⎯→A + Bn+ΔE = ΔE° − (RT/nF) ln ([A][Bn+]/[An+ ][B])n- number of e- transferred in rxnE°’ is the standard state reduction potential- reduction potential can be measured in half-cells connected by a wire and a saltbridge- standard state reduction potentials have been measured for most common redox rxnspositive ΔE means rxn is spontaneousΔG°' = −nFΔE°'NADH + FAD + H+ ⎯→⎯ NAD+ + FADH2FAD + 2H+ + 2e− ⎯→⎯ FADH2 E°' = −0.219VNADH ⎯→⎯ NAD+ + H+ + 2e− E°' = 0.315VE °’=-0.219E °’=0.315Δ E °’=0.096ΔG = −nFΔEΔG°’ = -2•96,485•0.096=-18.5kJ/molMethods to Elucidate Metabolic Pathways: 1. Interrupt the pathway (or perturb pathway) and look for changes in relative amounts of intermediates- Chemical Inhibitors that block the path- Mutations that inactivate specific enzymes2. Observe the transient passage of molecules through pathways- Heavy atom isotopes label nutrient with radioactive isotopes (eg. 14C, 32P) different isotopes can be used to probe different metabolic pathways(e.g. 32P to follow levels of phosphorylated compounds)- Real time observations with NMR -1D NMR spectrum can quantify relative amounts of different metabolites- gives characteristic chemical shifts for different compoundsmodern instruments can collect spectra on whole organisms-ex: observe glycogen levels in rat liver before and after injection with 13C glucose natural abundance 5 min after injection w/ 13C glucose observe accumulation of labeled glucose in liver observe α and β anomers 30 min after injections observe incorporation into glycogen3. high throughput