Complexities of the Reaction Mechanisms of CC Double Bond Reduction in Mammalian Fatty Acid Synthase Studied with Quantum Mechanics/Molecular Mechanics Calculations

摘要

Mammalian fatty acid synthase is a megaenzyme responsible for de novo fatty acid biosynthesis. NADPH-dependent enoyl reductase (ER) is one of its seven different catalytic domains. The "classical" reduction mechanism of C=C bonds catalysed by ER is postulated to take place through a hydride plus proton transfer to the substrate double bond. This mechanism was recently challenged because of the very unexpected experimental detection of two NADPH-substrate covalent adducts (A2 and A4) in enzymes similar to mammalian ER (mER). The fact that 16% of known enzymes used NADPH as a cofactor, mostly as a hydride donor, makes the discovery of previously unknown cofactor-substrate covalent intermediates very interesting. We studied the mechanism of reaction of ER using quantum mechanics/molecular mechanics (QM/MM) calculations, using three layers, two of them described by QM [the very accurate DLPNO-CCSD(T)/CBS and B3LYP/6-311+G(2d,2p)]. The rate limiting step of the classical pathway was the formation of an enolate intermediate upon hydride transfer (Delta G double dagger of 14.7 kcal. mol(-1), which is in very good agreement with the experimental value). Two alternative pathways, considering the recently detected A2 and A4 intermediates, were subsequently studied. The barriers for forming A2-wild type and A4-mutant (17.0 and 19.3 kcal.mol(-1)) also agreed very well with the experimental values. However, these species were incapable of forming the final product because of the very high Gibbs energy barriers to do so (>70 and >50 kcal.mol(-1)). They proved to be dead-end branches on the Gibbs energy surface, in chemical equilibrium with the intermediate enolate of the classical pathway. In summary, the classical reaction mechanism seems to hold in mER, but the discovery of unprecedented mechanistic hypotheses challenges the solidity and thoroughness with which we define and explore enzyme reaction mechanisms.