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

Mammalian fatty acid synthase is a mega-enzyme 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 (ΔG‡ 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.