“Lethal Synthesis” of Fluorocitrate by Citrate Synthase Explained through QM/MM Modeling

Abstract

The term “lethal synthesis” was coined for enzymatic formation of fluorocitrate, but this classic problem of enzyme stereoselectivity remains poorly understood. Here, we show that high-level ab initio quantum mechanics/ molecular mechanics (QM/MM) modeling can accurately capture this enzymatic enantioselectivity and the results provide detailed insight into its origin. Citrate synthase (CS) performs the first reaction in the citric acid cycle: the formation of citrate from oxaloacetate and acetate in the form of acetyl-CoA. When fluoroacetyl-CoA (from fluoroacetate) is used as a substrate instead of acetyl-CoA, 2fluorocitrate is formed, which inhibits aconitase, the next enzyme in the citric acid cycle. This process is responsible for the lethal toxicity of fluoroacetate to humans and other mammals. The fluorocitrate enantiomer that is predominantly formed by CS, (2R,3R)-fluorocitrate (Figure 1), is the same enantiomer that specifically inhibits aconitase. The only (semi-)quantitative experimental study published to date indicates that the minor product of the enzymatic formation of fluorocitrate, (2S,3R)-fluorocitrate, amounts to 2–3% of the major product. Using transition-state theory, this translates to a difference in activation free energy (DDG ) of 2.06– 2.30 kcalmol . The causes of this selectivity remain uncertain. We have previously modeled the two initial reaction steps for the natural substrates, proton abstraction from acetylCoA and condensation with oxaloacetate (OAA), in CS with high-level QM/MM methods. For the reaction with fluoroacetyl-CoA (FaCoA), the distinction between enantiomers is made in the proton-abstraction step, where either an Eor a Z-enolate is formed. Here, we show that 1) the calculated relative energy of the enolates accurately predicts the experimentally observed enantiospecificity and 2) the enantiospecificity is mostly due to the inherent energy difference of the reacting species. A model of the enzyme with OAA and FaCoA bound was built for QM/MM simulation, with the Asp375 side chain from Cb, the methylthioester part of FaCoA, and OAA in the QM region. QM/MM molecular dynamics indicated that preE and pre-Z conformations in the CS active site can be sampled in the same trajectory. Five different approximate transition-state conformations for both E-enolate and Zenolate formation were generated by QM/MM umbrella sampling molecular dynamics (see the Supporting Information). These conformations were used to perform high-level QM/MM modeling of proton abstraction, using the established reaction coordinate r= d(OAsp375H) d(CFaCoAH). Geometries were optimized at the B3LYP/6-31+G(d)//MM level and energies calculated at the SCS-MP2/aug-cc-pVDZ// MM level. This describes the reaction of CS with acetyl-CoA accurately, in agreement with local coupled-cluster QM/MM results. The QM/MM energy profiles for enolate formation show correctly that formation of the E-enolate is preferred (Figure 2). Boltzmann-weighted energy differences between the E and Z profiles are 1.80 and 2.05 kcalmol 1 for the activation energy and reaction energy in this step, respectively. Entropy contributions for Eand Z-enolate formation are expected to be (nearly) identical (supported by AM1/MM free energy profiles, see the Supporting Information). The Figure 1. Conversion of fluoracetyl-CoA to fluorocitrate by CS.