Biochemistry Chapter 14 Introduction to Metabolism Metabolism consists of anabolic and catabolic pathways Catabolism Anabolism Organisms classified by how they use energy degradative Usually energy yielding biosynthetic Usually energy requiring 1 carbon source 2 energy source autotrophs use CO2 as carbon source heterotrophs use C from organic source such as glucose phototrophs use light as energy source chemotrophs use oxidizable chemicals as energy source Role of O2 ultimate electron acceptor in oxidation of energy sources for large number of organisms aerobes ultimate e acceptor is O2 anaerobes 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 heterotrophs Vitamins and minerals are involved in metabolic reactions classified as water soluble or water insoluble water soluble usually converted into coenzymes participate 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 biosynthetic use ATP NADPH to produce reduced molecules and and NADPH NADP and ADP Organization 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 CO2 Metabolic pathways are compartmentalized cellular compartments cytosol glycolysis pentose phosphate pathway fatty acid biosynthesis some of gluconeogenesis mitochondria TCA cycle oxidative phosphorylation fatty acid oxidation amino acid breakdown smooth ER lipid and steroid biosynthesis expression of metabolic enzymes differ with different tissues liver is largely responsible for gluconeogenesis different tissues express different isozymes isozymes are enzymes with the same function but different kinetics or regulation Direction 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 G 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 G rxns with large negative G are at control points in pathways enzymes at key steps are regulated by allosteric control feedback inhibition products inhibit earlier rxns covalent modification phosphorylation dephosphorylation can change enzyme kinetics substrate cycling different enzymes for forward and reverse pathways can change reactant product ratios by changing reaction rates genetic control control transcription of metabolic enzymes Energy is stored shuttled between pathways via high energy compounds High energy compounds are often phosphorylated ATP high energy compound hydrolysis of phosphate has large negative G 3 major thermodynamic contributors to negative G 1 resonance stabilization of ATP is less than that of products 2 electrostatic repulsion of negative phosphates 3 smaller solvation energy of phosphoanhydride energy from high energy compounds used in coupled rxns use energy from high energy compound to give an initially unfavorable reaction a favorable G phosphocreatine high energy compound used 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 kinase ATP NDP ADP NTP other kinases generate NDPs from NTPs and NMPs e g adenylate kinase AMP ATP 2ADP Acetyl CoA high energy compound thioester bond is high energy intermediate in multiple pathways participates in acetyl group transfers boosts the energy to help rxns proceed Overall metabolic pathways involve redox reactions in catabolism move electrons from long energy rich compounds e g fats sugars to O2 to make CO2 and H2O and FAD The role of NAD 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 ATP Free energy of redox rxns described by Nernst equation relates redox potential to G E electrical potential difference measures likelihood of electrons transferring btw molecules in a redox rxn An B A Bn E E RT nF ln A Bn An B n number of e transferred in rxn E is the standard state reduction potential reduction potential can be measured in half cells connected by a wire and a salt bridge standard state reduction potentials have been measured for most common redox rxns positive E means rxn is spontaneous G nF E NADH FAD H NAD FADH2 FAD 2H 2e FADH2 E 0 219V NADH NAD H 2e E 0 315V E 0 219 E 0 315 E 0 096 G nF E G 2 96 485 0 096 18 5kJ mol Methods 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 enzymes 2 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 metabolites e g 32P to follow levels of phosphorylated compounds Real time observations with NMR 1D NMR spectrum can quantify relative amounts of different gives characteristic chemical shifts for different compounds modern 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 glycogen observe and anomers 30 min after injections observe incorporation into 3 high throughput data collection and processing has started new field of systems biology tries to quantify and model all interactions in a system at once metabolomics subset of systems biology dealing with metabolism allows for the characterization of complex pathways with multiple branches examine metabolites from blood urine etc quantify