Mechanism of the β-Ketoacyl Synthase Reaction Catalyzed by the Animal Fatty Acid Synthase

Abstract

The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form the beta-ketoacyl-S-acyl carrier protein product.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

“…Proposed KasA reaction mechanism. This mechanistic scheme is strongly supported by our structural work and builds on previous studies (13,44,54,55,57). Gray dashed lines indicate hydrogen bonds.…”

“…Based on our structural results concerning the binding mode of the malonyl group and taking previous findings into account (13,44,54,55), we propose the mechanism depicted in Fig. 8.…”

“…Subsequently, a similar outcome has been proposed and confirmed for Streptococcus pneumoniae FabF (spFabF) (55). Thus, an activated water molecule either directly attacks the carboxylate of malonyl-ACP or hydrates CO 2 after decarboxylation (13). The identification of a structured water molecule bound to the ⑀-nitrogen of His-311 (according to the KasA numbering) in most apo-KAS I/II crystal structures has previously been used to support this mechanism (55,57).…”

“…8. A mechanistic study of the mammalian fatty acid synthase ␤-ketoacyl synthase domain, which shares the Cys-His-His catalytic triad and other important active site residues with KAS I enzymes (56), revealed that bicarbonate is the product of the condensation reaction (13). Subsequently, a similar outcome has been proposed and confirmed for Streptococcus pneumoniae FabF (spFabF) (55).…”

“…During this process, the active site cysteine is acylated and subsequently attacked by the decarboxylated malonyl-AcpM to yield a ␤-ketoacyl-AcpM, which is extended by two carbon atoms. The acylated enzyme intermediate is mimicked by a C171Q KasA variant in which Gln-171 forms hydrogen bonds with the KasA oxyanion hole (11)(12)(13). This acylenzyme mimic is particularly important for future drug design studies as the natural products thiolactomycin (TLM) and platensimycin inhibit KAS enzymes by binding preferentially to the acyl-enzyme ( Fig.…”