page_title: Kinetic Isotope Effects of RuBisCO
section_title: Reaction details and energetics
hierarchical_section_title: Kinetic Isotope Effects of RuBisCO / Reaction details and energetics
caption_attribution_description: English: This figure shows the carboxylation of RuBP catalyzed by RuBisCO. Each step is shown in two panels: 1) The upper panel shows how each molecule is coordinated to the active site, while 2) The lower panel shows specifically how RuBP is being modified. Overall, the carboxylation of RuBP is a multi-step process.
context_page_description: The kinetic isotope effect of ribulose-1,5-bisphosphate carboxylase oxygenase is the isotopic fractionation associated solely with the step in the Calvin-Benson Cycle where a molecule of carbon dioxide is attached to the 5-carbon sugar ribulose-1,5-bisphosphate to produce two 3-carbon sugars called 3-phosphoglycerate. This chemical reaction is catalyzed by the enzyme RuBisCO, and this enzyme-catalyzed reaction creates the primary kinetic isotope effect of photosynthesis. It is also largely responsible for the isotopic compositions of photosynthetic organisms and the heterotrophs that eat them. Understanding the intrinsic KIE of RuBisCO is of interest to earth scientists, botanists, and ecologists because this isotopic biosignature can be used to reconstruct the evolution of photosynthesis and the rise of oxygen in the geologic record, reconstruct past evolutionary relationships and environmental conditions, and infer plant relationships and productivity in modern environments.
context_section_description: The fixation of CO
  2 by RuBisCO is a multi-step process. First, a CO
  2 molecule (that is not the CO
  2 molecule that is eventually fixed) attaches to the uncharged ε-amino group of lysine 201 in the active site to form a carbamate. This carbamate then binds to the magnesium ion (Mg²⁺) in RuBisCO's active site. A molecule of RuBP then binds to the Mg²⁺ ion. The bound RuBP then loses a proton to form a reactive, enodiolate species. The rate-limiting step of the Calvin-Benson Cycle is the addition of CO₂ to this 2,3-enediol form of RuBP. This is the stage where the intrinsic KIE of Rubisco occurs because a new C-C bond is formed. The newly formed 2-carboxy-3-keto-D-arabinitol 1,5-bisphophate molecule is then hydrated and cleaved to form two molecules of 3-phosphoglycerate (3 PGA). 3 PGA is then converted into hexoses to be used in the photosynthetic organism's central metabolism.
  The isotopic substitutions that can occur in this reaction are for carbon, oxygen, and/or hydrogen, though currently only a significant isotope effect is seen for carbon isotope substitution. Isotopes are atoms that have the same number of protons but varying numbers of neutrons. "Lighter" isotopes (like the stable Carbon-12 isotope) have a smaller overall mass, and "heavier" isotopes (like the stable Carbon-13 isotope or radioactive Carbon-14 isotope) have a larger overall mass. Stable isotope geochemistry is concerned with how varying chemical and physical processes preferentially enrich or deplete stable isotopes. Enzymes like RuBisCO cause isotopic fractionation because molecules containing lighter isotopes have higher zero-point energies (ZPE), the lowest possible quantum energy state for a given molecular arrangement. For this reaction, ¹³CO₂ has a lower ZPE than ¹²CO₂ and sits lower in the potential energy well of the reactants. When enzymes catalyze chemical reactions, the lighter isotope is preferentially selected because it has a lower activation energy and is thus more energetically favorable to overcome the high potential-energy transition state and proceed through the reaction. Here, ¹²CO₂ has a lower activation energy so more ¹²CO₂ than ¹³CO₂ goes through the reaction, resulting in the product (3 PGA) being lighter.
caption: Carboxylation of RuBP catalyzed by RuBisCO. Each step is shown in two panels: 1) The upper panel shows how each molecule is coordinated to the active site, while 2) The lower panel shows specifically how RuBP is being modified. Overall, the carboxylation of RuBP is a multi-step process.
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