No abstract

Large-scale conformational change is a common feature in the catalytic cycles of enzymes. Many enzymes function as homodimers with active sites that contain elements from both chains. Symmetric and anti-symmetric cooperative motions in homodimers can potentially lead to correlated active site opening and/or closure, likely to be important for ligand binding and release. Here, we examine such motions in two different domain-swapped homodimeric enzymes: the DcpS scavenger decapping enzyme and citrate synthase. We use and compare two types of all-atom simulations: conventional molecular dynamics simulations to identify physically meaningful conformational ensembles, and rapid geometric simulations of flexible motion, biased along normal mode directions, to identify relevant motions encoded in the protein structure. The results indicate that the opening/closure motions are intrinsic features of both unliganded enzymes. In DcpS, conformational change is dominated by an anti-symmetric cooperative motion, causing one active site to close as the other opens; however a symmetric motion is also significant. In CS, we identify that both symmetric (suggested by crystallography) and asymmetric motions are features of the protein structure, and as a result the behaviour in solution is largely non-cooperative. The agreement between two modelling approaches using very different levels of theory indicates that the behaviours are indeed intrinsic to the protein structures. Geometric simulations correctly identify and explore large amplitudes of motion, while molecular dynamics simulations indicate the ranges of motion that are energetically feasible. Together, the simulation approaches are able to reveal unexpected functionally relevant motions, and highlight differences between enzymes.

The term "lethal synthesis" was coined for enzymatic formation of fluorocitrate, [1] 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, 2-fluorocitrate is formed, [2] which inhibits aconitase, [3,4] the next enzyme in the citric acid cycle. This process is responsible for the lethal toxicity of fluoroacetate to humans and other mammals.[5] The fluorocitrate enantiomer that is predominantly formed by CS, (2R,3R)-fluorocitrate (Figure 1), is the same enantiomer that specifically inhibits aconitase.[4] 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.[6] Using transition-state theory, this translates to a difference in activation free energy (DDG°) of 2.06-2.30 kcal mol À1 . 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. [7][8][9] For the reaction with fluoroacetyl-CoA (FaCoA), the distinction between enantiomers is made in the proton-abstraction step, where either an E-or 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 pre-E 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(O Asp375 H)Àd(C FaCoA H). [7][8][9] 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. [8] The QM/MM energy profiles for enolate formation show correctly that formation of the E-enolate is preferred (Figure 2). Boltzmann-weighted energy differences b...

Intra class correlation coefficients between the countries were high: from 0.89 (England vs. US) through 0.99 (Canada vs. US). ConClusions: This proof of concept study indicates that computer-based choice tasks for the EQ-5D-5L in the general population are feasible and parameter of the choice tasks estimates are generally consistent and logical, and the estimated values are largely consistent between the 4 countries.

BackgroundA research register (Reach West) has been established to facilitate recruitment of people and patients to health-related research. We conducted a prospective feasibility study to investigate the practicality of recruiting through outpatient clinics.MethodsPatients over 18 years of age attending dental, eye or oncology outpatient clinics in an acute hospital in the West of England were provided with the opportunity to participate in Reach West. In Phase I, recruitment packs were handed to clinic attendees who could place completed consent forms in secure drop-box or return them later on-line or by post. In Phase II, recruitment packs were posted directly to patients with consent forms to be returned by post or on-line. Response rates by age, sex, postcode (for level of deprivation), and clinic type were recorded for those agreeing to participate on paper or on-line.ResultsIn Phase I, 2,314 of 4,500 (51.4%) of recruitment packs were handed out to clinic attendees, and 114 (5%) consented to join Reach West. In Phase II, 7,173 of 9000 packs were posted (79.7%), and 387 (5.4%) consented to participate. The overall consent rate was 6% (580), with the majority doing so on paper (87%) rather than on-line. The sample was balanced by sex, but mostly comprised people over 50 years located in less deprived postcodes. Non-staff costs for postal recruitment were lower than hand-outs in clinic (£6.84 compared with £8.05 per participant).ConclusionsRecruiting participants to the Reach West register was feasible among those with oncology, dental or eye outpatient appointments by post or with packs given out in the clinic. Response rates were similar to those achieved for other registers. Recruitment of participants can be achieved through outpatient clinics but other strategies will also be required to attract large numbers of participants and more diverse populations.

The term "lethal synthesis" was coined for enzymatic formation of fluorocitrate, [1] 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, 2-fluorocitrate is formed, [2] which inhibits aconitase, [3,4] the next enzyme in the citric acid cycle. This process is responsible for the lethal toxicity of fluoroacetate to humans and other mammals.[5] The fluorocitrate enantiomer that is predominantly formed by CS, (2R,3R)-fluorocitrate (Figure 1), is the same enantiomer that specifically inhibits aconitase.[4] 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.[6] Using transition-state theory, this translates to a difference in activation free energy (DDG°) of 2.06-2.30 kcal mol À1 . 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. [7][8][9] For the reaction with fluoroacetyl-CoA (FaCoA), the distinction between enantiomers is made in the proton-abstraction step, where either an E-or 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 pre-E 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(O Asp375 H)Àd(C FaCoA H). [7][8][9] 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. [8] The QM/MM energy profiles for enolate formation show correctly that formation of the E-enolate is preferred (Figure 2). Boltzmann-weighted energy differences b...

No abstract