Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase Domain of Human Fatty Acid Synthase

Human fatty acid
synthase (hFAS) is a large multienzyme that catalyzes
all steps of fatty acid synthesis, which is overexpressed in many
cancer cells. Studies have shown that FAS inhibitors exhibit antitumor
activity without relevant effects over normal cells. Therefore, the
molecular description of active sites in hFAS should stimulate the
development of inhibitors as anticancer drug candidates. The malonyl-acetyl
transferase (MAT) domain is responsible for loading acetyl-CoA and
malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a
carrier for fatty acid reaction intermediates. In this work, we have
applied computational QM/MM methods at the DLPNO–CCSD­(T)/CBS:AMBER
level of theory to study the MAT reaction mechanism. The results indicate
that the initial catalytic stage occurs in two sequential steps: (1)
nucleophilic attack on the thioester carbonyl group of the substrate
through a concerted pathway that involves a Ser-His dyad and (2) tetrahedral
intermediate breakdown and release of the free coenzyme A. The Gibbs
activation energies for the first and second steps are 13.0 and 6.4
kcal·mol–1 and 10.9 and 8.0 kcal·mol–1, whether the substrate transferred to the MAT domain
was acetyl-CoA or malonyl-CoA, respectively. Both Met499 and Leu582
form an oxyanion hole that lodges the negative charge of the substrate
carbonyl, lowering the first step energetic barriers for both substrates.
The mutation of the Arg606 residue by an alanine severely impairs
the malonyl transfer reaction, while leading to a kinetic improvement
of the transferase activity for acetyl-CoA, which is in agreement
with earlier experimental studies. The results from this work encourage
future studies that aim for the full comprehension of the MAT catalytic
reaction and for the rational design of novel antineoplastic drugs
that target this domain.