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General Metabolism II Andy Howard Introductory Biochemistry, fall 2010 18 November 2010 Biochemistry: Metabolism II 11/18/2010 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 … but first, we need to explain the role of Ca2+ in muscle contraction Biochemistry: Metabolism II 11/18/2010 Page 2 of 47 What we’ll discuss Metabolism Control Feedback Flux Phosphorylation Other PTMs Evolution Redox Tools for studying Biochemistry: Metabolism II Nutrition Macronutrients Proteins Fats Carbohydrates Vitamins Fat-soluble Water-soluble 11/18/2010 Page 3 of 47 iClicker quiz question 1 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 II 11/18/2010 Page 4 of 47 iClicker quiz question 2 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 II 11/18/2010 Page 5 of 47 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 II 11/18/2010 Page 6 of 47 Metabolic flux, illustrated Courtesy Jeremy Zucker’s wiki http://bio.freelogy.org/wiki/User:JeremyZucker#Metabolic_Engineering_tutorial Biochemistry: Metabolism II 11/18/2010 Page 7 of 47 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 II 11/18/2010 Page 8 of 47 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 II 11/18/2010 Page 9 of 47 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 II 11/18/2010 Page 10 of 47 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 II 11/18/2010 Page 11 of 47 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 II 11/18/2010 Page 12 of 47 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 II Muscle phosphorylase EC 2.4.1.1 192kDa dimer monomer shown PDB 2GJ4, 1.6Å 11/18/2010 Page 13 of 47 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 II 11/18/2010 Page 14 of 47 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 II 11/18/2010 Page 15 of 47 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 II 11/18/2010 ADP-ribosylation of arginine; fig. courtesy RPI Page 16 of 47 Metabolism and evolution Metabolic pathways have evolved over hundreds of millions of years to work efficiently and with appropriate controls Biochemistry: Metabolism II 11/18/2010 Page 17 of 47 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 II 11/18/2010 Page 18 of 47 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 II 11/18/2010 Page 19 of 47 Evolving a branch Original pathway: D E1 E2 A B C E3 X Fully evolved pathway: E3a D ABC E3b X Biochemistry: Metabolism II 11/18/2010 Page 20 of 47 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 II 11/18/2010 Page 21 of 47 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 II 11/18/2010 Page 22 of 47 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 II 11/18/2010 Page 23 of 47 Oxidation-reduction reactions and Energy Oxidation-reduction reactions involve transfer of electrons, often along with other things Generally compounds with many C-H bonds are high in energy because the carbons can be oxidized (can lose electrons) Biochemistry: Metabolism II 11/18/2010 Page 24 of 47 Reduction potential Reduction potential is a measure of thermodynamic activity in the context of movement of electrons Described in terms of half-reactions Each half-reaction has an electrical potential, measured in volts, associated with it because we can (in principle) measure it in an electrochemical cell Biochemistry: Metabolism II 11/18/2010 Page 25 of 47 So what is voltage, anyway? Electrical potential is available energy per unit charge: 1 volt = 1 Joule per coulomb 1 coulomb = 6.24*1018 electrons Therefore energy is equal to the potential multiplied by the number of electrons Biochemistry: Metabolism II 11/18/2010 Page 26 of 47 Electrical potential and energy This can be expressed thus: Go’ = -nFEo’ n is the number of electrons transferred F = fancy way of writing # of Coulombs (which is how we measure charge) in a mole (which is how we calibrate our energies) = 96.48 kJ V-1mol-1 Biochemistry: Metabolism II 11/18/2010 Page 27 of 47 Oh yeah? Yes. 1 mole of electrons = 6.022 * 1023 e1 coulomb = 6.24*1018 e1 mole = 9.648*104 Coulomb 1 V = 1 J / Coulomb=10-3 kJ / Coulomb Therefore the energy per mole associated with one volt is 10-3 kJ / C * 9.648*104 C = 96.48 kJ Biochemistry: Metabolism II 11/18/2010 Page 28 of 47 What can we do with that? The relevant voltage is the difference in standard reduction potential between two half-reactions Eo’ = Eo’acceptor - Eo’donor Combined with free energy calc, we see Eo’ = (RT/nF ) lnKeq and E = Eo’ - (RT/nF ) ln [products]/[reactants] This is the Nernst equation Biochemistry: Metabolism II 11/18/2010 Page 29 of 47 Free energy from electron transfer We can examine tables of electrochemical half-reactions to get an idea of the yield or requirement for energy in redox reactions Example: NADH + (1/2)O2 + H+ -> NAD+ + H2O; We can break that up into half-reactions to determine the energies Biochemistry: Metabolism II 11/18/2010 Page 30 of 47 Half-reactions and energy NAD+ + 2H+ + 2e- NADH + H+, Eo’ = -0.32V (1/2)O2 + 2H+ + 2e- H2O, Eo’ = 0.82V Reverse the first reaction and add: NADH + (1/2)O2 + H+ NAD+ + H2O, Eo’ = 0.82+0.32V = 1.14 V. Go’ = -nFEo’ = -2*(96.48 kJ V-1mol-1)(1.14V) = -220 kJ mol-1; that’s a lot! Biochemistry: Metabolism II 11/18/2010 Page 31 of 47 Absorbance How to detect NAD reactions NAD+ 340 nm NADH NAD+ and NADH (and NADP+ and NADPH) Wavelength have extended aromatic systems But the nicotinamide ring absorbs strongly at 340 only in the reduced (NADH, NADPH) forms Spectrum is almost pH-independent, too! So we can monitor NAD and NADPdependent reactions by appearance or disappearance of absorption at 340 nm Biochemistry: Metabolism II 11/18/2010 Page 32 of 47 Classical metabolism studies Add substrate to a prep and look for intermediates and end products If substrate is radiolabeled (3H, 14C) it’s easier, but even nonradioactive isotopes can be used for mass spectrometry and NMR NMR on protons, 13C, 15N, 31P Reproduce reactions using isolated substrates and enzymes Biochemistry: Metabolism II 11/18/2010 Page 33 of 47 Next level of sophistication… Look at metabolite concentrations in intact cell or organism under relevant physiological conditions Note that Km is often ~ [S]. If that isn’t true, maybe you’re looking at the non-physiological substrate! Think about what’s really present in the cell. Biochemistry: Metabolism II 11/18/2010 Page 34 of 47 Mutations in single genes If we observe or create a mutation in a single gene of an organism, we can find out what the effects on viability and metabolism are In humans we can observe genetic diseases and tease out the defective gene and its protein or tRNA product Sometimes there are compensating enzyme systems that take over when one enzyme is dead or operating incorrectly Biochemistry: Metabolism II 11/18/2010 Page 35 of 47 Deliberate manipulations Bacteria and yeast: Irradiation or exposure to chemical mutagens Site-directed mutagenesis Higher organisms: We can delete or nullify some genes; thus knockout mice Introduce inhibitors to pathways and see what accumulates and what fails to be synthesized Biochemistry: Metabolism II 11/18/2010 Page 36 of 47 Nutrition Lots of nonsense, some sense on this subject Skepticism among MDs as to its relevance Fair view is that nutrition matters in many conditions, but it’s not the only determinant of health Biochemistry: Metabolism II 11/18/2010 Page 37 of 47 Macronutrients Proteins Carbohydrates Lipids Fiber Biochemistry: Metabolism II 11/18/2010 Page 38 of 47 Protein as food Source of essential amino acids Source of non-essential aa Fuel (often via interconversion to ketoacids and incorporation into TCA) All of the essential amino acids must be supplied in adequate quantities Biochemistry: Metabolism II 11/18/2010 Page 39 of 47 Which amino acids are essential? At one level, that’s an easy question to answer: they’re the ones for which we lack a biosynthetic pathway: KMTVLIFWH That shifts the question to: why have some of those pathways survived and not all? Answer: pathways that are complex or require more than ~30 ATP / aa are absent (except R,Y) Biochemistry: Metabolism II 11/18/2010 Page 40 of 47 The human list AA Asp Asn Lys Met Thr Ala Val Leu Ile moles ATP 21 22-24 50-51 44 31 20 39 47 55 essential? no no yes yes yes no yes yes yes Glu Gln 30 31 no no Biochemistry: Metabolism II AA moles ATP Arg 44 Pro 39 Ser 18 Gly 12 Cys 19 Phe 65 Tyr 62 Trp 78 His 42 11/18/2010 essential? no no no no no yes no* yes yes Page 41 of 47 Carbohydrates as food Generally recommended to be more than half of caloric intake Complex carbohydrates are hydrolyzed to glucose-1-P and stored as glycogen or interconverted into other metabolites Biochemistry: Metabolism II 11/18/2010 Page 42 of 47 Lipids as food You’ll see in 402 that the energy content of a lipid is ~ 2x that of carbohydrates simply because they’re more reduced They’re also more efficient food storage entities than carbs because they don’t require as much water around them Certain fatty acids are not synthesizable; by convention we don’t call those vitamins Biochemistry: Metabolism II 11/18/2010 Page 43 of 47 Vitamins Vitamins are necessary micronutrients A molecule that is a vitamin in one organism isn’t necessarily a vitamin in another E.coli can make all necessary metabolites given sources of water, nitrogen, and carbon Most eukaryotic chemoautotrophs find it more efficient to rely on diet to make complex metabolites We’ll discuss lipid vitamins first, then water-soluble vitamins Biochemistry: Metabolism II 11/18/2010 Page 44 of 47 Why wouldn’t organisms make everything? Complex metabolites require energy for synthesis Control of their synthesis is also metabolically expensive Cheaper in the long run to derive these nutrients from diet Biochemistry: Metabolism II 11/18/2010 Page 45 of 47 Vitamins: broad classifications Water-soluble vitamins Coenzymes or coenzyme precursors Non-coenzymic metabolites Fat-soluble vitamins Antioxidants Other lipidic vitamins Biochemistry: Metabolism II 11/18/2010 Page 46 of 47 Are all nutrients that we can’t synthesize considered vitamins? No: If it’s required in large quantities, it’s not a vitamin By convention, essential fatty acids like linoleate aren’t considered vitamins Biochemistry: Metabolism II 11/18/2010 Page 47 of 47
