Abstract

Dehydratase (DH) is a catalytic domain of the mammalian fatty acid synthase (mFAS), a multidomain enzyme with seven different active sites that work in tandem to carry out the biosynthesis of palmitic acid for de novo lipogenesis. DH catalyzes the dehydration of the beta-hydroxyacyl to an alpha,beta-unsaturated acyl intermediate. We have conducted hybrid QM/MM calculations to clarify the catalytic mechanism for the DH domain at the ONIOM(DFT/Amber) level of theory. The results have shown that the dehydration step occurs in two stages: (i) the His878-imidazole acts as a base deprotonating the C-alpha of the beta-hydroxyacyl (HAC) substrate and (ii) the beta-elimination of the beta-hydroxyl of HAC proceeds with late protonation of the leaving hydroxide by the Asp1033-carboxylic group, forming a water molecule as a byproduct. The a-deprotonation depends on an oxyanion hole mechanism where the HAC's alpha-carbonyl is anchored by two strong hydrogen bonds from the neighboring G1y888 and the intramolecular beta-hydroxyl, positioning the C-alpha of HAC for deprotonation by His878. A positively charged His1037 improves the acidic character of Asp1033 and completes the catalytic triad in DH, because when His1037 is neutral the positively charged His878 behaves as the acid in the fi-elimination step. We observe that the positively charged His1037 renders the beta-elimination step more thermodynamically favorable (Delta(r)G of 15.9 kcal center dot mol(-1)). The beta-elimination step exhibits a Gibbs energy barrier of 14.1 kcal center dot mol(-1) and it is the rate-limiting step of the reaction (in agreement with the experimental barrier of similar to 17 kcal center dot mol(-1). Nevertheless, the rate-limiting step does not seem to be dependent on the protonation of His1037. Through evaluation of the electrostatic effect per residue on the rate-limiting step, we concluded also that the electrostatic contribution of the enzyme's body does not seem significant, even though there are many positively and negatively charged residues close to the leaving beta-hydroxyl group of HAC.