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GE 140a 2019 Lecture 13 Organismal/system scale kinetic isotope effects

GE 140a 2019 Lecture 13 Organismal/system scale kinetic isotope effects. The broadest overview of biosynthetic isotopic fractionation. These sorts of reactions involve r eduction of inorganic carbon (or analogous c hanges in oxidation state and bonding e nvironment of H, N, S, etc.)

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GE 140a 2019 Lecture 13 Organismal/system scale kinetic isotope effects

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  1. GE 140a 2019 Lecture 13 Organismal/system scale kinetic isotope effects

  2. The broadest overview of biosynthetic isotopic fractionation These sorts of reactions involve reduction of inorganic carbon (or analogous changes in oxidation state and bonding environment of H, N, S, etc.) and are generally highly fractionating “You are what you eat, give or take a per mil” principle: Bulk living biomass of heterotrophs is similar in d13C to their food; i.e., If you ‘shuffle’ reduced carbon instead of reducing oxidized carbon, there is little isotopic discrimination

  3. The two major complications And yet, the net isotope effects of similar forms of autotrophic fixation can be highly variable And molecular-scale isotopic diversity within one organism can be tremendous. This suggests very large, diverse elementary isotope effects, which are masked at the organismal level by ‘mean reversion’ of all these wildly different isotopic pools 13C/12C

  4. Some major forms of metabolic transformations of carbon CO2 in air 0.0112 Photosynthesis C4 plants 0.0110 Abundance of 13C Photosynthesis C3 plants 0.0108 Fermentation/ CO2 reduction Microbial methane 0.0106

  5. Biosynthetic fractionations: Concepts and governing eqns. See Hayes, 2001, RiM volume for detailed explanation di: d13CPDB value of reservoir i d#: d13CPDB value of material transferred along reaction path # f#: Flux (in moles of carbon) along reaction path # e#: Isotopic discrimination associated with reaction path #, calculated as –1000.(aproduct-reactant - 1) (e.g., e2 = 25 means d2 is ca. 25 ‰ less than dB). Steady state at a given site: Sf#d# = 0 and Sf# = 0 (not applied to non-exchangeable input and output reservoirs, e.g., A, D and G and I above)

  6. Steady state at point B If A is an infinite reservoir, dA is a constant If A is a finite reservoir, RA = RAinitial.F(a1-1) d1 ~ dA-e1 d2 ~ dB-e2 f1 = f2 at steady state d1f1 ~ d2f2 at steady state Ergo, d1 ~ d2 at steady state (mass balance at site B) Ergo, dB =dA-e1+e2 at steady state For our example, dB = dA+25 ‰

  7. C3 photosynthesis: the canonical carbon isotope reaction network da, Ca: set by atmosphere d1 = da - et d2 = di - ef d3 = di - et For a steady state Ci: f1 - f2 - f3 = 0 Isotopic steady state of di: f1d1 - f2d2 - f3d3 = 0 Define ƒ2 = f2/f1 Ergo, d1 = ƒ2d2 + (1-ƒ2)d3 And, ep = da - df = ef -ƒ2(ef - et) et: diffusive fractionation; usually 4.4 ‰ ef: metabolic fractionation; usually 27 ‰

  8. C4 photosynthesis: move the show into aqueous solution [CO2]aq [CO2]atm Decarboxylation of Oxaloacetate ef~ 27 ‰ etw= diffusion through water; ~ 0.8 ‰ Leakage from site of decarboxylation L = f5/f4 ep4 = eta + [ec - eb/d + L(ef - etw) - eta](1-ƒ2) eta = diffusive fractionation (ca. 4.4 ‰ usually) etw = diffusion in water; ~ 0.8 ‰ ec ~ 2.2 ‰ eb/d set by CO2-HCO3- equilibrium; usually -6 ‰ ef = 27 ‰ CO2 uptake site

  9. Hayes, 2001, Reviews in Mineralogy vol. 43

  10. Empirical model for marine phytoplankton photosynthetic fractionation ep = -182 x [(µ x V)/(ce x A)] + 25.3

  11. A common form of carbon isotope forensics Tracking the diets of migratory elephants Cerling et al., 2006 Cerling et al., 2006

  12. Isotopic proof that Floyd Landis is both a cheat and not very smart Isotopic composition of Floyd Landis’ hormones C4 plants (e.g., corn) 0.01107 0.01101 13C abundance All the boring ones 0.01095 His massively elevated testosterone Synthetic testosterone is usually made from phytosterolpercursors, typically from soy C3 plants (e.g., soy bean)

  13. Finally, let’s return to the problem of large molecular-scale isotopic diversity This must reflect, in some fashion, the strong energetic differences between isotope effects at structurally non-equivalent molecular sites

  14. KIE’s are generally strongly position specific in vitro pyruvate decarboxylation Pyruvate 20 Observed 10 (‰) 0 KIE ~25 ‰ -10 d13C (C2) — d13C (C1) -20 Low d13C -30 Equilibrium Low d13C C1 High d13C C2 -40 CO2 10 20 30 40 T (˚C) Acetyl group DeNiro and Epstein, 1977

  15. Position specificity is often ’passed’ from precursor to product, meaning isotopic structures of organics are a record of several steps in their biosynthetic pathways Lipid synthesis inherits the acetyl group ‘fingerprint’ CO2 KIE ~25 ‰ ‘Elongation’ Low d13C Pyruvate Acetyl group High d13C “C=O” C=O C=O C=O C=O C=O C=O C=O “CH3” CH3 CH3 CH3 CH3 CH3 CH3 CH3 d13C DeNiro and Epstein, 1977; Monson and Hayes, 1982

  16. Part of the complexity on molecule- and site-specific isotopic diversity arises from the fact that even ‘elementary’ enzymatic reactions can unfold in different ways in different contexts Michaelis-Menten kinetics Here the net rate law approaches ‘zero order’ Rate Here the net rate law has some normal order ≥1 Reactant (‘substrate’) concentration Reactant concentration Fastest rate observed Vmax x [S] Rate = KM + [S] Concentration at which rate is half of Vmax

  17. This is because an ‘elementary’ enzymatic reaction is in fact its own little reaction network that functions like a 0-dimensional flux model ef Vmax = kcatx [Enzyme]0 Kf, ef Kcat, ecat Enzyme bound to substrate Enzyme + free substrate Enzyme + product kb, eb • A common default assumption is that free and bound substrate exist in a reversible equilibrium (rate forward and back of that first step are balanced), and transformation to product is irreversible • But this is an end member and over simplification; the system works more like our box model for C3 photosynthesis, where each of these half reactions is fractionating, bound substrate is at a ‘branch point’ that chooses between back reaction or transformation to product, and the branching ratio will dictate how strongly the enzymatic a is expressed • And, many reactions that seem ‘elementary’ are in fact concerted chains of reactions involving multiple enzymes, substrates and operations. Thus, an enzymatic reaction can work sort of like our box model for C4 photosynthesis • It is also possible for the final transformation to product to be partially reversible

  18. And, connecting back to our discussion of elementary kinetics in lecture 12, the ‘job’ of an enzyme is lower the activation energy barrier, which it does by manipulating the transition state structure. Thus, the true, fundamental KIE (‘ecat’) is a function of enzymatic structure, which is subject to evolutionary diversification Non catalyzed transition state Catalyzed transition state Substrate (reactant) Product Reaction coordinate

  19. A biosynthetic reaction involving several mostly reversible steps: Methanogenesis from CO2 Note all steps up to methyl Coenzyme M are reversible; we should expect these will (more or less) conform to local equilibrium. If you know enough about structure, these can be understood through the Urey-Bigeleisen theory you already know.

  20. A biosynthetic reaction involving several mostly reversible steps: Methanogenesis from CO2 More ‘reversible’ with less overstepping of H addition steps CO2 CO2 HC-R HC-R ∆Grxn = = O O   R R HC HC Reaction progress H2C=R H2C=R ∆Grxn H3C-R H3C-R CH4 CH4 Valentine, 2004; Stolper et al., 2015

  21. A biosynthetic reaction involving several mostly reversible steps: Methanogenesis from CO2 This model is supported by the fact that microbial methane formed at low growth rates conforms to thermodynamic equilibrium for isotopic clumping properties 7 N=5 Ni equilibration Projected endmember 6 Pyrolysis In-place thermogenic (Hanesville) 5 Sub-surface biogenic (Gulf of Mexico, Santa Barbara basin, Antrim shale) Theory 13CH3D enrichment 4 (‰) 3 2 1 0 0 100 200 300 400 500 600 700 Temperature (˚C)

  22. A fun ‘wild card’: maybe metabolic networks act to equilibrate molecular isotopic contents and structures Galimov, 1985; 2006

  23. Using stable isotopes to construct budgets of carbon in the surface environment and geological record

  24. Simplified view of 13C budget used in most models of rock record d13C = -6 ‰ Net deposition of carbonate Net deposition of reduced C ∆13Ccarb-org Mantle input = + (implies size of ocean/atmosphere/biosphere inventory does not change)

  25. d13Cmantle input = fcarbd13Ccarb + forgd13Corg fcarb + forg = 1 d13Cmantle input = -6.0 ‰, PDB d13Ccarb - d13Corg ~ 30 ‰ Forg ~ (d13Ccarb + 6)/30 ~ 0.2 + (d13Ccarb/30)

  26. An isotopic budget for carbon

  27. Phanerozoic record of avg. marine carbonate d13C P/T extinction Antarctic glaciation begins Paleozoic extinctions and radiations K/T extinction Carboniferous coal swamps Period of persistent glaciations Ripperdan, 2001, Reviews in Mineralogy vol. 43

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