Theoretical and Experimental Determination on Two Substrates Turned over by 4-Oxalocrotonate Tautomerase

• Published in: The journal of physical chemistry. A, Molecules, spectroscopy, kinetics, environment, & general theory Molecules, spectroscopy, kinetics, environment, & general theory, 2006-01, Vol.110 (2), p.700-708
• Main Authors: Cisneros, G. Andrés, Wang, Min, Silinski, Peter, Fitzgerald, Michael C, Yang, Weitao
• Format: Article
• Language: English
• Subjects: Isomerases - metabolism; Kinetics; Models, Molecular; Models, Theoretical; Protons; Sorbic Acid - analogs & derivatives; Sorbic Acid - metabolism; Substrate Specificity
• ISSN: 1089-5639; 1520-5215
• DOI: 10.1021/jp0543328

Quantum mechanical/molecular mechanical (QM/MM) calculations and experimental kinetic studies have been performed on 4-oxalocrotonate tautomerase (4OT) for two different substrates, 2-hydroxymuconate (2HM) and 2-oxo-4-hexenedioate (2o4hex). Potential (ΔE) and free energy (ΔG) paths for both steps of the reaction using both substrates were calculated to determine the free energy barriers and compared to the experimental values obtained from the kinetic studies via the transition state theory. In the first step, a proton from the hydroxyl oxygen on the second carbon of 2HM, or from the third carbon of 2o4hex, is abstracted by Pro-1. In the second step, the proton is transferred to the fifth carbon of the substrate to form the product, 2-oxo-3-hexenedioate (2o3hex). For both substrates we obtain a calculated ΔG of ≈ 13 kcal/mol, in agreement with experimental determinations. The calculated free energy barrier difference ΔG 2o4 hex − ΔG 2 HM (ΔΔG) is 0.87 kcal/mol. We obtained an experimental ΔΔG of 0.85 kcal/mol. These results suggest that 2HM is turned over faster than 2o4hex by 4OT. However, these energy differences are so small that both 2HM and 2o4hex need to be taken into account in considering the mechanism of catalysis of 4OT.