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Regulation of Metabolic Pathways 1. Metabolic pathways 2. Basic enzyme kinetics 3. Metabolic pathway regulation Basic Functions of Metabolic Pathways Classification of Reactions ! Fueling reactions (catabolic pathways) » Produce precursor metabolites needed for biosynthesis » Generate energy (ATP) for cellular functions » Produce reducing power (NAPDH) for biosynthesis ! Biosynthetic reactions (biosynthetic pathways) » Produce building blocks for macromolecular synthesis » Produce coenzymes & signaling molecules ! Polymerization reactions » Form macromolecules from building blocks ! Assembly reactions » Chemical modifications of macromolecules to form cellular structures (cell wall, membranes) http://www.expasy.org/cgi-bin/show_thumbnails.pl Membrane Transport Processes ! Free diffusion » Species transported down concentration gradient » Transport driven by chemical potential difference ! Facilitated diffusion » Species transported down concentration gradient » Specific carrier or transmembrane protein involved ! Active transport » Species can be transported up concentration gradient » Specific proteins or permeases involved Catabolic Pathways ! Basic functions » Generate energy & reducing power » Produce precursors for biosynthesis ! Participating pathways » » » » » » Glycolytic pathway Pentose phosphate pathway (PPP) Fermentative pathways Tricarboxylic acid (TCA) cycle Anaplerotic pathways Pathways involved in catabolism of fats, organic acids & amino acids Glycolytic Pathway ! Also called the Embden-Meyerhof-Parnas (EMP) pathway ! Converts glucose to pyruvate ! Generates ATP & NADH ! ! Glucose + 2 NAD+ + 2 ADP + 2 Pi " 2 pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H20 Also produces three precursors for biosynthesis » Glyceraldehyde 3-phosphate (G3P) » 3-phoshoglycerate (3-PG) » Phosphoenolpyruvate (PEP) Simplified Picture of Glycolytic Pathway Pentose Phosphate Pathway (PPP) ! Converts G6P to NAPDH & two precursors for biosynthesis » Ribose-5-phosphate (R5P) » Erythrose-4-phosphate (E4P) ! ! Also produces glycolytic intermediates F6P & G3P Relative flux through EMP & PPP varies » Energy & reducing power requirements » Need for precursor metabolites ! PPP stoichiometry depends on extent carbon is recycled back to EMP Oxidative PPP Non-Oxidative PPP Overall PPP Fermentative Pathways ! Fermentation » Occurs under oxygen limited conditions » Pyruvate converted into metabolic products (lactic acid, acetic acid, ethanol) ! Bacteria » Several different metabolic products formed » Mixed acid fermentation ! Yeasts » Ethanol is the main metabolic product » Alcohol fermentation » Limited acetate & succinate also formed Yeast Fermentation TCA Cycle ! ! ! Also called the citric acid cycle or Krebs cycle Completely oxidizes pyruvate to produce ATP via oxidative phosphorylation Location » Bacteria – occurs in cytosol » Yeast – occurs in mitochondria ! Stoichiometry » Pyruvate + CoA + NAD " Acetyl-CoA + CO2 + NADH + H+ » Acetyl-CoA + 3 NAD + FAD + GDP + Pi + 2H20 " 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA TCA Cycle in Yeast Simplified Picture of TCA Cycle Oxidative Phosphorylation in Yeast ! Basic functions » Regenerate NAD+ for glycolysis » Generate ATP for biosynthesis ! Basic mechanism » Electrons are transported from NADH & FADH through the electron transport chain to oxygen » Electron transport causes protons to be released into the intermembrane space » These electrons can be transported back into mitochondrial matrix by a proton conducting ATP-synthase » The detailed mechanistic steps are not completely understood ! Theoretical yields » P/O ratio: 3 ATP/NADH & 2 ATP/FADH » Overall: 15 ATP/pyruvate » Actual yields are lower due to incomplete coupling of the oxidative & phosphorylation processes Oxidative Phosphorylation Biosynthetic Pathways ! ! Generate 12 precursor metabolites needed for cellular synthesis Amino acid biosynthesis » Forms 20 common amino acids » Well characterized in bacteria & yeast » Consumes considerable energy & reducing power ! Biosynthesis of other building blocks » » » » Nucleotides " RNA & DNA Fatty acids " lipids UDP-glucose " storage carbohydrates Also consume energy & reducing power Metabolic Pathway Regulation ! Basic concepts » Pathways must be regulated to compensate for changes in nutrient availability & cellular demands » Regulated variables include concentrations of substrate, enzyme, product & special regulatory molecules » Regulation implemented over a very wide range of time scales (15 orders of magnitude) ! Present focus » Regulation of enzyme activity at relatively short time scales » Best understood form of metabolic regulation Hierarchy of Regulatory Mechanisms Time Scale of Regulatory Mechanisms Enzyme Regulation ! Regulation of enzyme activity » Achieved at the metabolic level » Feedback inhibition & activation of enzyme activity by pathway substrate/products or global metabolites (ATP, NADH) » Fast responses (second time scale) ! Regulation of enzyme concentration » » » » Achieved at the gene level Repression & induction of enzyme synthesis Slow responses (hour time scale) Focus of lecture on signal transduction networks Michaelis-Menten Kinetics Reversible Inhibition ! Substrate inhibition ! » Rate inhibited by high substrate concentrations v" ! » Inhibitor reversibly binds to enzyme on non-active vmax sites S regulatory v" vmax S Km ! S ! S 2 / Ki 1 ! I / Ki K m ! S Competitive inhibition » Inhibitor competes with substrate for enzymatic active sites vmax S v" K m (1 ! I / K i ) ! S Non-competitive inhibition ! Uncompetitive inhibition » Inhibitor vmax reversibly S binds to v" enzyme-substrate K m complex 1! I / K i 1 ! I / Ki !S Allosteric Enzymes ! Cooperativity » Binding to one vacant site induces altered affinities for remaining vacant sites » Homotrophic – only substrate involved » Heterotrophic – involve substrate & regulator ! Allosteric enzymes » » » » Exhibit cooperativity Composed of multiple catalyic & regulatory subunits Characterized by sigmoidal velocity curves Allow large change in reaction rate for small changes in substrate concentration » Facilitate regulation of metabolic pathways where the substrate concentrations exhibit small variations Cooperative Binding ! Types of cooperative effects » Positive – binding of first molecule activates binding of second molecule » Negative – binding of first molecule inhibits binding of second molecule vmax S n v" Hill equation K ! Sn n " number of sites Glycolytic Pathway Regulation of EMP & PPP in Yeast ! Glycolytic (EMP) pathway » Hexokinase: inhibited by G6P (product inhibition) » Phosphofructokinase – Inhibited by ATP & citrate (signals overabundance of TCA cycle intermediates) – Activated by AMP & ADP (signal lack of available energy) – Activated by ammonia, phosphate & fructose-2,6bisphosphate (regulatory molecule) » Pyruvate kinase: inhibited by ATP & acetyl-CoA ! Pentose phosphate pathway » Glucose-6-phosphate dehydrogenase: activity regulated by NAPDH/NADP+ ratio Glycolytic Regulation in Yeast Phosphofructokinase (PFK) Regulation ! PFK is a complex allosteric enzyme » Inhibited by ATP » Activated by fructose-2,6-bisphosphate (F-2,6-P) » F-2,6-P formed by phosphorylation of F6P by ATP ! Cooperativity » ATP binding decreases affinity for F6P (substrate) » F-2,6-P binding causes large increase in F6P affinity » Glycolytic flux stimulated by F-2,6-P & inhibited by ATP ! Regulatory effects » Allows PFK to increase activity in response to increasing F6P concentration » High energy levels suppress PFK activity TCA Cycle in Yeast Regulation of the TCA Cycle in Yeast ! Citrate synthase » Weakly inhibited by NADH/NAD+ ratio ! Isocitrate dehydrogenase » Strongly inhibited by NADH/NAD+ ratio » Activated by AMP » Inhibited by ATP ! Alpha-ketoglutarate dehydrogenase » Weakly inhibited by NADH/NAD+ ratio Regulation at the Gene Level Summary ! Metabolic pathways » Classified according to their function » Focused on yeast central carbon metabolism ! Metabolic pathway regulation » Essential to control metabolic function » Implemented over a wide range of time scales » Focused on short-term regulation of enzymatic activities » Can involve complex mechanisms ! Implications » Complexity necessitates integrative approaches » Metabolic pathway modeling & analysis References ! ! ! ! G. H. Braus, ”Aromatic Amino Biosynthesis in the Yeast Saccharomyces cerevisiae: a Model System for the Regulation of a Eukaroytic Biosynthetic Pathway,” Microbiological Reviews, 55, 349-370 (1991). J. Nielsen & J. Villadsen, Bioreaction Engineering Principles, Plenum Press, New York, NY (1994). M. L. Shuler & F. Kargi, Bioprocess Engineering: Basic Concepts, 2nd edition, Prentice Hall, Englewood Cliffs, NJ (2002). G. N. Stephanopoulos, A. A. Aristidou & J. Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press, New York, NY (1998).