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General Metabolism I Andy Howard Introductory Biochemistry 24 November 2009 Biochemistry: Metabolism I 11/24/2009 Metabolism: the core of biochem All of biology 402 will concern itself with the specific pathways of metabolism Our purpose here is to arm you with the necessary weaponry Biochemistry: Metabolism I 11/24/2009 Page 2 of 75 What we’ll discuss Metabolism Definitions Pathways Control Feedback Phosphorylation Thermodynamics Kinetics Biochemistry: Metabolism I Cofactors Tightly-bound metal ions as cofactors Activator ions as cofactors Cosubstrates Prosthetic groups 11/24/2009 Page 3 of 75 Metabolism Almost ready to start the specifics (chapter 18) Define it! Metabolism is the network of chemical reactions that occur in biological systems, including the ways in which they are controlled. So it covers most of what we do here! Biochemistry: Metabolism I 11/24/2009 Page 4 of 75 Intermediary Metabolism Metabolism involving small molecules Describing it this way is a matter of perspective: Do the small molecules exist to give the proteins something to do, or do the proteins exist to get the metabolites interconverted? Biochemistry: Metabolism I 11/24/2009 Page 5 of 75 How similar are pathways in various organisms? Enormous degree of similarity in the general metabolic approaches all the way from E.coli to elephants Glycolysis arose prior to oxygenation of the atmosphere This is considered strong evidence that all living organisms are derived from a common ancestor Biochemistry: Metabolism I 11/24/2009 Page 6 of 75 Anabolism and catabolism Anabolism: synthesis of complex molecules from simpler ones Generally energy-requiring Involved in making small molecules and macromolecules Catabolism: degradation of large molecules into simpler ones Generally energy-yielding All the sources had to come from somewhere Biochemistry: Metabolism I 11/24/2009 Page 7 of 75 Common metabolic themes Maintenance of internal concentrations of ions, metabolites, & (? enzymes) Extraction of energy from external sources Pathways specified genetically Organisms & cells interact with their environment Constant degradation & synthesis of metabolites and macromolecules to produce steady state Biochemistry: Metabolism I 11/24/2009 Page 8 of 75 Metabolism and energy Biochemistry: Metabolism I 11/24/2009 Page 9 of 75 Metabolic classifications Carbon sources Autotrophs vs. heterotrophs Atmospheric CO2 as a C source vs. otherwise-derived C sources Energy sources Phototrophs vs. chemotrophs (Sun)light as source of energy vs. reduced organic compounds as a source of energy Biochemistry: Metabolism I 11/24/2009 Page 10 of 75 Fourway divisions (table 17.2) Energy/Carbon Phototrophs: Energy from light Chemotrophs: Energy from reduced organic molecules Autotrophs: Carbon from atmospheric CO2 Photoautotrophs: Green plants, cyanobacteria, … Chemoautotrophs: Nitrifying bacteria, H, S, Fe bacteria Heterotrophs: Photoheterotrophs: Chemoheterotrophs: Carbon from other Nonsulfur purple Animals, many [organic] sources bacteria microorganisms, . . . Biochemistry: Metabolism I 11/24/2009 Page 11 of 75 Another distinction: the organism and oxygen Aerobes: use O2 as the ultimate electron acceptor in oxidation-reduction reactions Anaerobes: don’t depend on O2 Obligate: poisoned by O2 Facultative: can switch hit Biochemistry: Metabolism I 11/24/2009 Page 12 of 75 Flow of energy Sun is ultimate source of energy Photoautotrophs drive synthesis of [reduced] organic compounds from atmospheric CO2 and water Chemoheterotrophs use those compounds as energy sources & carbon; CO2 returned to atmosphere Biochemistry: Metabolism I 11/24/2009 Page 13 of 75 How to anabolism & catabolism interact? Sometimes anabolism & catabolism occur simultaneously. How do cells avoid futile cycling? Just-in-time metabolism Compartmentalization: Anabolism often cytosolic Catabolism often mitochondrial Biochemistry: Metabolism I 11/24/2009 Page 14 of 75 Pathway A sequence of reactions such that the product of one is the substrate for the next Similar to an organic synthesis scheme (but with better yields!) May be: Unbranched Branched Circular Biochemistry: Metabolism I 11/24/2009 Page 15 of 75 Catabolism stages Stage 1: big nutrient macromolecules hydrolyzed into their building blocks Stage 2: Building blocks degraded into limited set of simpler intermediates, notably acetyl CoA Stage 3: Simple intermediates are fed to TCA cycle and oxidative phosphorylation Biochemistry: Metabolism I 11/24/2009 Page 16 of 75 Anabolism stages Short list of simple precursors These are elaborated in characteristic ways to build monomers e.g.: transamination of -ketoacids to make -amino acids Those are then polymerized to form proteins, polysaccharides, polynucleotides, etc. Biochemistry: Metabolism I 11/24/2009 Page 17 of 75 Some intermediates play two roles Some metabolites play roles in both kinds of pathways We describe them as amphibolic Just recall that: catabolism is many down to few, anabolism is few up to many Biochemistry: Metabolism I 11/24/2009 Page 18 of 75 Differences between catabolic and anabolic pathways Often they share many reactions, notably the ones that are nearly isoergic (G ~ 0) Reactions with G < -20 kJ mol-1 are not reversible as is Those must be replaced by (de)coupled reactions so that the oppositely-signed reactions aren’t unfeasible Biochemistry: Metabolism I 11/24/2009 Page 19 of 75 Other differences involve regulation Generally control mechanisms influence catalysis in both directions Therefore a controlling influence (e.g. an allosteric effector) will up- or down-regulate both directions If that’s not what the cell needs, it will need asymmetric pathways or pathways involving different enzymes in the two directions Biochemistry: Metabolism I 11/24/2009 Page 20 of 75 ATP’s role We’ve discussed its significance as an energy currency It’s one of two energy-rich products of the conversion of light energy into chemical energy in phototrophs ATP then provides drivers for almost everything else other than redox Biochemistry: Metabolism I 11/24/2009 Page 21 of 75 NAD’s role QuickTime™ and a decompressor are needed to see this picture. NAD acts as as an electron acceptor via net Image courtesy Michigan Tech transfer of hydride ions, Biological Sciences H:-, in catabolic reactions Reduced substrates get oxidized in the process, and their reducing power ends up in NADH Energy implied by that is used to make ATP (3.5 ATP/NAD) in oxidative phosphorylation Biochemistry: Metabolism I 11/24/2009 Page 22 of 75 NADPH’s role Involved in anabolic redox reactions Reducing power in NADPH NADP used to reduce some organic molecule Involves hydride transfers again NADPH regenerated in phototrophs via light-dependent reactions that pull electrons from water Biochemistry: Metabolism I 11/24/2009 Page 23 of 75 How do we study pathways? Inhibitor studies Mutagenesis Isotopic traces (radio- or not) NMR Disruption of cells to examine which reactions take place in which organelle Biochemistry: Metabolism I 11/24/2009 Page 24 of 75 Why multistep pathways? Limited reaction specificity of enzymes Control of energy input and output: Break big inputs into ATP-sized inputs Break energy output into pieces that can be readily used elsewhere Biochemistry: Metabolism I 11/24/2009 Page 25 of 75 iClicker quiz question 1 A reaction A+B C+D proceeds from left to right in the cytosol and from right to left in the mitochondrion. As written, it is probably (a) a catabolic reaction (b) an anabolic reaction (c) an amphibolic reaction (d) we don’t have enough information to answer. Biochemistry: Metabolism I 11/24/2009 Page 26 of 75 iClicker quiz question 2 An asymmetry between stage 1 of catabolism (C1) and the final stage of anabolism (A3) is (a) A3 always requires light energy; C1 doesn’t (b) A3 never produces nucleotides; C1 can involve nucleotide breakdown (c) A3 adds one building block at a time to the end of the growing polymer; C1 can involve hydrolysis in the middle of the polymer (d) There are no asymmetries between A3 and C1 Biochemistry: Metabolism I 11/24/2009 Page 27 of 75 iClicker quiz question 3 Could dAMP, derived from degradation of DNA, serve as a building block to make NADP? (a) Yes. (b) Probably not: the energetics wouldn’t allow it. (c) Probably not: the missing 2’-OH would make it difficult to build NADP (d) No: dAMP is never present in the cell Biochemistry: Metabolism I 11/24/2009 Page 28 of 75 Regulation Organisms respond to change Fastest: small ions move in msec Metabolites: 0.1-5 sec Enzymes: minutes to days Flow of metabolites is flux: steady state is like a leaky bucket Addition of new material replaces the material that leaks out the bottom Biochemistry: Metabolism I 11/24/2009 Page 29 of 75 Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial Biochemistry: Metabolism I 11/24/2009 Page 30 of 75 Feedback and Feed-forward Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway Biochemistry: Metabolism I 11/24/2009 Page 31 of 75 Feedback realities Control usually exerted at first committed step (i.e., the first reaction that is unique to the pathway) Controlling element is usually the last element in the path Often the controlled reaction has a large negative Go’. Biochemistry: Metabolism I 11/24/2009 Page 32 of 75 Feed-forward Early metabolite activates a reaction farther down the pathway Has the potential for instabilities, just as in electrical feed-forward Usually modulated by feedback Biochemistry: Metabolism I 11/24/2009 Page 33 of 75 Activation and inactivation by post-translational modification Most common: covalent phosphorylation of protein usually S, T, Y, sometimes H Kinases add phosphate Protein-OH + ATP Protein-O-P + ADP … ATP is source of energy and Pi Phosphatases hydrolyze phosphoester: Protein-O-P +H2O Protein-OH + Pi … no external energy source required Biochemistry: Metabolism I 11/24/2009 Page 34 of 75 Phosphorylation’s effects Phosphorylation of an enzyme can either activate it or deactivate it Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated Example: glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme Biochemistry: Metabolism I 11/24/2009 Page 35 of 75 Glycogen phosphorylase Reaction: extracts 1 glucose unit from non-reducing end of glycogen & phosphorylates it: (glycogen)n + Pi (glycogen)n-1 + glucose-1-P Activated by phosphorylation via phosphorylase kinase Deactivated by dephosphorylation by phosphorylase phosphatase Biochemistry: Metabolism I 11/24/2009 Page 36 of 75 Amplification Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins Thus a single activation event at the kinase level can trigger many events at the target level Biochemistry: Metabolism I 11/24/2009 Page 37 of 75 Other PTMs Are there other reversible posttranslational modifications that regulate enzyme activity? Yes: Adenylation of Y ADP-ribosylation of R Uridylylation of Y Oxidation of cysteine pairs to cystine Cis-trans isomerization of prolines Biochemistry: Metabolism I 11/24/2009 Page 38 of 75 Evolution of Pathways: How have new pathways evolved? Add a step to an existing pathway Evolve a branch on an existing pathway Backward evolution Duplication of existing pathway to create related reactions Reversing an entire pathway Biochemistry: Metabolism I 11/24/2009 Page 39 of 75 Adding a step E1 E2 E3 E4 E5 ABCDEP Original pathway • When the organism makes lots of E, there’s good reason to evolve an enzyme E5 to make P from E. • This is how asn and gln pathways (from asp & glu) work Biochemistry: Metabolism I 11/24/2009 Page 40 of 75 Evolving a branch Original pathway: D E1 E2 A B C E3 X Fully evolved pathway: E3a D ABC E3b X Biochemistry: Metabolism I 11/24/2009 Page 41 of 75 Backward evolution Original system has lots of E P E gets depleted over time; Then D gets depleted; need to make it from D, so we evolve enzyme E4 to do that. need to make it from C, so we evolve E3 to do that And so on Biochemistry: Metabolism I 11/24/2009 Page 42 of 75 Duplicated pathways Homologous enzymes catalyze related reactions; this is how trp and his biosynthesis enzymes seem to have evolved Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination) Biochemistry: Metabolism I 11/24/2009 Page 43 of 75 Reversing a pathway We’d like to think that lots of pathways are fully reversible Usually at least one step in any pathway is irreversible (Go’ < -15 kJ mol-1) Say CD is irreversible so E3 only works in the forward direction Then D + ATP C + ADP + Pi allows us to reverse that one step with help The other steps can be in common This is how glycolysis evolved from gluconeogenesis Biochemistry: Metabolism I 11/24/2009 Page 44 of 75 Many cofactors are derived from vitamins We justify lumping these two topics together because many cofactors are vitamins or are metabolites of vitamins. Biochemistry: Metabolism I 11/24/2009 Page 45 of 75 Family tree of cofactors Cofactors, coenzymes, essential ions, cosubstrates, prosthetic groups: Cofactors (apoenzyme + cofactor holoenzyme) Coenzymes Essential ions Activator ions (loosely bound) Ions in metalloenzymes Cosubstrates (loosely bound) Biochemistry: Metabolism I 11/24/2009 Prosthetic groups (tightly bound) Page 46 of 75 Metal-activated enzymes Absolute requirements for mobile ions Often require K+, Ca2+, Mg2+ Example: Kinases: Mg-ATP complex Metalloenzymes: firmly bound metal ions in active site Usually divalent or more Sometimes 1e- redox changes in metal Biochemistry: Metabolism I 11/24/2009 Page 47 of 75 Coenzymes Organic moeities that enable enzymes to perform their function: they supply functionalities not available from amino acid side chains Cosubstrates Enter reaction, get altered, leave Repeated recycling within cell or organelle Prosthetic groups Remain bound to enzyme throughout Change during one phase of reaction, eventually get restored to starting state Biochemistry: Metabolism I 11/24/2009 Page 48 of 75 Major cosubstrates Facilitate group transfers, mostly small groups Oxidation-reduction participants Cosubstrate ATP S-adenosylMet UDP-glucose NAD,NADP Coenzyme A Tetrahydrofolate Ubiquinone Source Function Transfer P,Nucleotide Methyl transfer Glycosyl transfer Niacin 2-electron redox Pantothenate Acyl transfer Folate 1Carbon transfer Lipid-soluble e- carrier Biochemistry: Metabolism I 11/24/2009 Page 49 of 75 Major prosthetic groups Transfer of larger groups One- or two-electron redox changes Prosth.gp. FMN, FAD TPP PLP Biotin Adenosylcobalamin MeCobal. Lipoamide Retinal Vitamin K Source Riboflavin Thiamine Pyridoxine Biotin Cobalamin Function 1e- and 2e- redox transfers 2-Carbon transfers with C=O Amino acid group transfers Carboxylation, COO- transfer Intramolec. rearrangements Cobalamin Methyl-group transfers Transfer from TPP Vision Carboxylation of glu residues Vitamin A Vitamin K Biochemistry: Metabolism I 11/24/2009 Page 50 of 75 Adenosine triphosphate Synthesizable in liver (chapter 18) Building block for RNA Participates in phosphoryl-group transfer in kinases Source of other coenzymes Biochemistry: Metabolism I 11/24/2009 Page 51 of 75 S-adenosylmethionine Made from methionine and adenosine Sulfonium group is highly reactive: can donate methyl groups Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University Biochemistry: Metabolism I 11/24/2009 Page 52 of 75 UDP-glucose Most common donor of glucose Formed via: Glucose-1P + UTPUDP-glucose + PPi Reaction driven to right by PPi hydrolysis Structure courtesy of UIC Pharmacy Program Biochemistry: Metabolism I 11/24/2009 Page 53 of 75 NAD+ and NADP+ Net charge isn’t really >0 ; the + is just a reminder that the nicotinamide ring is positively charged Most important cosubstrates in oxidationreduction reactions in aerobic organisms Structure courtesy of Sergio Marchesini, U. Brescia Biochemistry: Metabolism I 11/24/2009 Page 54 of 75 Differences between them The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety The functional difference is that NAD is usually associated with catabolic reactions and NADP is usually associated with anabolic reactions Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products Exceptions: photosynthesis and ETC! Biochemistry: Metabolism I 11/24/2009 Page 55 of 75 How do we get back to the starting point? NADH is often oxidized back to NAD+ as part of the electron-transport chain Imbalances can be addressed via NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase Biochemistry: Metabolism I 11/24/2009 Page 56 of 75 iClicker quiz question 4 Based on what you have learned, would you expect glycogen synthase to be activated or inhibited by phosphorylation? (a) activated (b) inhibited (c) neither (d) insufficient information to tell Biochemistry: Metabolism I 11/24/2009 Page 57 of 75 iClicker quiz question 5 What would you expect to be the phosphate donor in the NAD kinase reaction? (a) free phosphate (b) pyrophosphate (c) ATP (d) pyridoxal phosphate Biochemistry: Metabolism I 11/24/2009 Page 58 of 75 Reduced forms of NAD(P) Reduction occurs on the nicotinamide ring Ring is no longer netpositive Ring is still planar but the two hydrogens on the para carbon are not Biochemistry: Metabolism I 11/24/2009 Page 59 of 75 FAD and FMN Flavin group based on riboflavin Alternate participants in redox reactions Prosthetic groups: tightly but noncovalently bound to their enzymes That protects against wasteful reoxidation of reduced forms FADH2 is weaker reducing agent than NADH These are capable of one-electron oxidations and reductions Biochemistry: Metabolism I 11/24/2009 Page 60 of 75 FAD and FMN structures FAD has an AMP attached P to P Structure courtesy Paisley University Biochemistry: Metabolism I 11/24/2009 Page 61 of 75 FMN/FAD redox forms Two-electron version: H+ + :H- transferred Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University Biochemistry: Metabolism I 11/24/2009 Page 62 of 75 (ADP-3’P) Coenzyme A Reactive portion is free sulfhydryl at one end of the molecule Can form thioester with acetate, etc. Pantoate + b-alanine = pantothenate Biochemistry: Metabolism I (Pantoate) 2-mercaptoethylamine) b-alanine) Structure courtesy of MPB project, George Washington University 11/24/2009 Page 63 of 75 Thiamine Pyrophosphate Based on thiamine, vitamin B1 Carboxylases and oxidative decarboxylases use this coenzyme So do transketolases (move 2 carbons at a time between sugars with keto groups) Thiazolium ring is reactive center: pKa drops from 15 in H2O to 6 in enzyme Biochemistry: Metabolism I 11/24/2009 Page 64 of 75 TPP reactions pyrimidine thiazolium Diagram courtesy of Oklahoma State U. Biochemistry program Biochemistry: Metabolism I 11/24/2009 Page 65 of 75 Pyridoxal phosphate PLP is prosthetic group for many amino-acid-related enzymes, particularly transaminations Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine Biochemistry: Metabolism I 11/24/2009 Page 66 of 75 Biotin Rarity: vitamin is the prosthetic group Used in reactions that transfer carboxyl groups … and in ATP-dependent carboxylations Biochemistry: Metabolism I 11/24/2009 Page 67 of 75 Biotin reactivity Covalently bound to active-site lysines to form species called biocytin Pyruvate carboxylase is characteristic reaction: Diagram courtesy University of Virginia Biochemistry Biochemistry: Metabolism I 11/24/2009 Page 68 of 75 Tetrahydrofolate Primary donor of one-carbon units (formyl, methylene, methyl) Supplies methyl group for thymidylate Dihydrofolate reductase (DHFR) is an interesting drug target Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells Trimethoprim as antibacterial: Bacterial DHFR is somewhat different from eucaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate Biochemistry: Metabolism I 11/24/2009 Page 69 of 75 THF structure and function Figure courtesy horticulture program, Purdue Biochemistry: Metabolism I 11/24/2009 Page 70 of 75 Cobalamin Largest B vitamin Structure related to heme but missing one carbon in ring structure Cobalt bound in core of ring system Involved in enzymatic rearrangements Catabolism of odd-chain fatty acids Methylation of homocysteine Reductive dehalogenation Biochemistry: Metabolism I 11/24/2009 Page 71 of 75 AdenosylCobalamin Reactive Co-C bond “Missing” carbon Diagram courtesy of Swiss Food News Biochemistry: Metabolism I 11/24/2009 Page 72 of 75 Lipoamide Protein-bound form of lipoic acid Contains five-membered disulfide ring Covalently bound via amide to protein lysine sidechain Involved in swinging arm between active sites in multienzyme complexes Disulfides break periodically Example: pyruvate dehydrogenase complex Biochemistry: Metabolism I 11/24/2009 Page 73 of 75 Lipoamide 2e- reduction Cf. Scheme 7.6: thioester starting point Fig. Courtesy Biochem and Biophysics program, Rensselaer Biochemistry: Metabolism I 11/24/2009 Page 74 of 75 iClicker quiz question 6 Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate aspartate + -ketoglutarate? (a) ascorbate (b) PLP ( c) thiamine pyrophosphate (d) NAD (e) none of the above Biochemistry: Metabolism I 11/24/2009 Page 75 of 75