The fixation of  by RuBisCO is a multi-step process. First, a  molecule (that is not the  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 (Mg) in RuBisCOs active site. A molecule of RuBP then binds to the Mg 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 CO 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-bisphosphate 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 organisms 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, CO has a lower ZPE than CO 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, CO has a lower activation energy so more CO than CO goes through the reaction, resulting in the product (3 PGA) being lighter.