The alanine transaminase (ALT) enzyme catalyzes the transfer of an amino group from alanine to α-ketoglutarate to produce pyruvate and glutamate. Isotope fractionation factors (IFFs) for the reaction ⁺H₃NCH(CH₃)COO⁻ + ⁻OOCCH₂CH₂C(O)COO⁻ ↔ CH₃C(O)COO⁻ + ⁻H₃NCH(CH₂CH₂COO⁻)COO⁻ (zwitterionic neutral alanine + doubly deprotonated α-ketoglutarate ↔ pyruvate + zwitterionic glutamate anion) were calculated from the partition functions of explicitly and implicitly solvated molecules at 298 K. Calculations were done for alanine (non-charge separated, zwitterion, deprotonated,), pyruvic acid (neutral, deprotonated), glutamic acid (non-charge separated, zwitterion, deprotonated, doubly deprotonated), and α-ketoglutaric acid (neutral, deprotonated, doubly deprotonated). The computational results, calculated from gas phase and aqueous optimized clusters with explicit H₂O molecules at the MP₂/aug-cc-pVDZ and MP₂/aug-cc-pVDZ/COSMO levels, respectively, predict that substitution of ¹³C at the C2 position of alanine and pyruvic acid and their various forms leads to the C2 position of pyruvic acid/pyruvate being enriched in ¹³C/¹²C ratio by 9 ‰. Simpler approaches that estimate the IFFs based solely on changes in the zero-point energies (ZPEs) are consistent with the higher-level model. ZPE-based IFFs calculated for simple analogues formaldehyde and methylamine (analogous to the C₂ positions of pyruvate and alanine, respectively) predict a ¹³C enrichment in formaldehyde of 7 to 8 ‰ at the MP₂/aug-cc-pVDZ and aug-cc-pVTZ levels. A simple predictive model using canonical functional group frequencies and reduced masses for ¹³C exchange between R₂C=O and R₂CH-NH₂ predicted enrichment in R₂C=O that is too large by a factor of two, but is qualitatively accurate compared with the more sophisticated models. Our models are all in agreement with the expectation that pyruvate and formaldehyde will be preferentially enriched in ¹³C due to the strength of their >C=O bond relative to that of the ≡C-NH₂ in alanine and methylamine. ¹³C/¹²C substitution is also modeled at the methyl and carboxylic acid sites of alanine and pyruvic acid, respectively
