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BMED 3510 Metabolic Systems Book Chapter 8 What is Metabolism? Etymology: Greek “meta · ballein” ~ to throw about, to change Metabolism is the set of life-sustaining chemical transformations within cells of living organisms. Metabolites are the intermediates and products of metabolism. The term ''metabolite'' is usually restricted to small molecules. A primary metabolite is directly involved in processes of normal growth, development, and reproduction (e.g. glucose and pyruvate). A secondary metabolite is not directly involved in those processes, but usually has an important ecological function. Examples include antibiotics and pigments. http://www.news-medical.net/; en.wikipedia.org What is Metabolomics? Metabolomics is the scientific study of biochemical systems involving large numbers of metabolites at the same time. Metabolomics is the systematic study of the unique chemical fingerprints that specific cellular processes leave behind. Currently there are ≈42,000 metabolites in HMDB2, of which: 22,000 are associated with a proteins (enzyme & transporters) 5,000 have been quantified. en.wikipedia.org & www.hmdq.ca Problem Assessing Metabolites The metabolome unlike the genome or the proteome is chemically very diverse. Metabolites can be water-soluble, lipid-soluble, gases, organic/inorganic, positively/negatively charged (sol)… Wide concentration range: from the molar range down to nothing. Present in several compartments (cytosol, mitochondrial, ER, ect). Half-lives of metabolites are extremely variable, with some metabolites being very short-lived inside cells Assessing Metabolites Nature Protocols 6, 1241–1249 (2011) Quenching in Cold Methanol Nature Protocols 6, 1241–1249 (2011) Detection Http://www.bruker.com Metabolic networks Http://www.infohow.org Thermodynamics • Spontaneous reactions only occur between high and low energy metabolites. (Reactions w/ negative ΔG) • Achieved by: Coupled reactions. Prentice Hall c2002 What are Enzymes? Proteins that catalyze (bio)chemical reactions Why do we Have Enzymes? • Higher reaction rates • Most reactions don’t even occur spontaneously. • Greater reaction specificity • • • Absolute Relative (group) Stereospecificity (Stereoisomers) • Milder reaction conditions • Capacity for regulation COO - COO • Metabolites have many potential pathways of conversion or decomposition NH2 O OH COO OH COO Chorismate mutase COO OOC O NH2 - - O COO COO OH - • Enzymes allow substrate to be channeled into the product of highest demand In the Absence of an Enzyme S P In the Presence of an Enzyme S ES EP P How is ∆G Lowered? Catalytic Cycle of an Enzyme 1 Substrates enter active site; enzyme changes shape so its active site embraces the substrates (induced fit). Substrates Enzyme-substrate complex 6 Active site Is available for two new substrate Mole. Enzyme 5 Products are Released. Figure 8.17 Products 2 Substrates held in active site by weak interactions, such as hydrogen bonds and ionic bonds. 3 Active site (and R groups of its amino acids) can lower EA and speed up a reaction by • acting as a template for substrate orientation, • stressing the substrates and stabilizing the transition state, • providing a favorable microenvironment, • participating directly in the catalytic reaction. 4 Substrates are Converted into Products. Enzyme Kinetics • • Thermodynamics: Energy and possibility of a reaction Kinetics: Speed of a reaction • • Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by – Enzyme – Substrate & Products – Effectors (Inhibitors & Activators) – Environmental conditions (Temperature, pH) Measuring Enzyme Kinetics Kinetic Properties of Enzymes Study of the effect of substrate concentration on the rate of reaction Henri-Michaelis-Menten Kinetics Adian Brown (1902) Victor Henri (1903) Archibald Vivian Hill (1910) Michaelis & Menten (1913) Briggs & Aldane (1925) E + S k1 k-1 E S k2 E S k1 S E k1 ( ES ) ( E S ) k1 S E (k1 k2 ) ( ES ) P k 2 ( ES ) + P Henri-Michaelis-Menten Kinetics Assumptions: S k1 S E k1 ( ES ) ( E S ) k1 S E (k1 k2 ) ( ES ) P k ( ES ) Concentrations 2 • Reaction mechanism • Homogeneous (well stirred) • k1, k-1 >> k2 • S >> ET (ET=E+ES) P • Quasi-Steady-State Assumption: S ( ES ) 0 ES Time Henri-Michaelis-Menten Kinetics ( E S ) k1 S E (k1 k2 ) ( ES ) 0 k1 S E (k1 k2 ) ( ES ) ( ES ) k1 S E S E k 1 k 2 Km K m ( ES ) S ( ET ES ) ( ES ) S ET Km S S k 2 ET S Vmax v P k 2 ( ES ) Km S Km S Km k 1 k 2 k1 ET E ES P k 2 ( ES ) Vmax k2 ET Henri-Michaelis-Menten Kinetics Reaction speed Vmax vP Vmax S vp KM S Vmax/2 KM [S] Henri-Michaelis-Menten Kinetics • KM is the apparent dissociation constant of the ES complex and measures the enzyme’s affinity for the substrate. - KM values of enzymes differ widely. - KM provides approximation of substrate concentration in vivo. - For most enzymes, KM lies between 10-1 and 10-7M. - High KM indicates weak binding. - Low KM indicates strong binding. • Vmax = kcat ET • kcat (k2) is the “turnover number”: measures how many substrate molecules one enzyme molecule converts per unit of time. • ET is the total enzyme concentration - Most enzymes are not saturated with substrate - kcat/KM can be used as a measure of catalytic efficiency. - Diffusion limits the catalytic efficiency. Why? kcat/KM < K1 Hill Kinetics Reactions catalyzed by enzymes with n subunits Reaction speed Vmax vP n Vmax S vp n KM S n Vmax/2 KM [S] Common Enzymatic Mechanisms Enzyme Inhibition Inhibitors are compounds that decrease the enzyme activity • Irreversible inhibitors (inactivators) react with the enzyme - one inhibitor molecule can permanently shut off one enzyme molecule - they are often powerful toxins but also may be used as drugs • Reversible inhibitors bind to, and can dissociate from the enzyme - they may be structural analogs of substrates or products - they are often used as drugs to slow down a specific enzyme • Competitive inhibition: the inhibitor competes with the substrate for the active site. -Can be overcome by a sufficiently high concentration of substrate. -Inhibitor increase the Km value. • Allosteric inhibition: the inhibitor binds to a different binding site. Competitive Inhibition Vmax S v S K m 1 I KI KM is modulated Uncompetitive & Noncompetitive Effects of Inhibitors Effects of Inhibitors http://employees.csbsju.edu/hjakubowski/classes/ch331/transkinetics/olinhibition.html http://www.as.wvu.edu/~rbrundage/lecture2b/img011.gif Databases: BRENDA Databases: KEGG Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Pathway Regulation Negative feedback • Product of a pathway controls the rate of synthesis by inhibiting an early step, usually the first “committed” step (unique to the pathway). Positive feedforward • Metabolite early in the pathway activates an enzyme further down the pathway Glycolysis in L. lactis A.R. Neves et al. FEMS Microbiol. Rev. 29 (2005) 531–554 Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Amino Acid Biosynthesis http://www.uky.edu/~dhild/biochem/24/lect24.html Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Reversible Covalent Modification Reversible Covalent Modification Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Insulin-Induced Glucose Uptake Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Multifunctional Enzymes Metabolic Regulation How is metabolism regulated? • Enzymatic regulation: activation or Inhibition (comp., uncomp.) • Pathway regulation: - Negative feedback - Positive feedforward • Isoenzymes, two or more proteins that catalyze the same reaction - Different kinetics, regulatory properties and cellular distribution - Coded by different genes or by alternative splicing • Reversible covalent modification • Compartmentalization • Multienzyme complexes and multifunctional enzymes - Metabolite channeling - “channeling” of reactants between active sites • Transcription, translation, mRNA and protein degradation Summary Advantages of having enzyme catalyzed reactions How do enzymes operate? Michaelis-Menten rate law (approximation) Effect of different types of inhibition Metabolic regulation many modes, operating at different time scales