Reaction Energetics and 13C Fractionation of Alanine Transamination in the Aqueous and Gas Phases

The alanine transaminase enzyme catalyzes the transfer of an amino group from alanine to a-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 alpha-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 (noncharge separated, zwitterion, deprotonated), pyruvic acid (neutral, deprotonated), glutamic acid (noncharge separated, zwitterion, deprotonated, doubly deprotonated), and a-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 C-13 at the C-2 position of alanine and pyruvic acid and their various forms leads to the C-2 position of pyruvic acid/pyruvate being enriched in C-13/C-12 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 C-2 positions of pyruvate and alanine, respectively) predict a 13C enrichment in formaldehyde of 7-8% at the MP2/aug-cc-pVDZ and aug-cc-pVTZ levels. A simple predictive model using canonical functional group frequencies and reduced masses for C-13 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 because of the strength of their >C=O bond relative to that of C-NH2 in alanine and methylamine. C-13/C-12 substitution is also modeled at the methyl and carboxylic acid sites of alanine and pyruvic acid, respectively.