title: Substrate polarization in enzyme catalysis: QM/MM analysis of the effect of oxaloacetate polarization on acetyl-CoA enolization in citrate synthase.
abstract: Citrate synthase is an archetypal carbon-carbon bond forming enzyme. It promotes the conversion of oxaloacetate (OAA) to citrate by catalyzing the deprotonation (enolization) of acetyl-CoA, followed by nucleophilic attack of the enolate form of this substrate on OAA to form a citryl-CoA intermediate and subsequent hydrolysis. OAA is strongly bound to the active site and its alpha-carbonyl group is polarized. This polarization has been demonstrated spectroscopically, [(Kurz et al., Biochemistry 1985;24:452-457; Kurz and Drysdale, Biochemistry 1987;26:2623-2627)] and has been suggested to be an important catalytic strategy. Substrate polarization is believed to be important in many enzymes. The first step, formation of the acetyl-CoA enolate intermediate, is thought to be rate-limiting in the mesophilic (pig/chicken) enzyme. We have examined the effects of substrate polarization on this key step using quantum mechanical/molecular mechanical (QM/MM) methods. Free energy profiles have been calculated by AM1/CHARMM27 umbrella sampling molecular dynamics (MD) simulations, together with potential energy profiles. To study the influence of OAA polarization, profiles were calculated with different polarization of the OAA alpha-carbonyl group. The results indicate that OAA polarization influences catalysis only marginally but has a larger effect on intermediate stabilization. Different levels of treatment of OAA are compared (MM or QM), and its polarization in the protein and in water analyzed at the B3LYP/6-31+G(d)/CHARMM27 level. Analysis of stabilization by individual residues shows that the enzyme mainly stabilizes the enolate intermediate (not the transition state) through electrostatic (including hydrogen bond) interactions: these contribute much more than polarization of OAA.
authors.affiliation: Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, United Kingdom.
authors.forename: Marc W
authors.initials: MW
authors.lastname: van der Kamp
authors.forename: Francesca
authors.initials: F
authors.lastname: Perruccio
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Mulholland
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D002645:Chickens; D002950:Citrate (si)-Synthase / Q000737:chemistry*; D004563:Electrochemistry; D005740:Gases / Q000737:chemistry; D006860:Hydrogen Bonding; D008958:Models, Molecular; D062907:Oxaloacetic Acid / Q000737:chemistry*; D011522:Protons; D011789:Quantum Theory; D012996:Solutions; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: D005740:Gases; D011522:Protons; D012996:Solutions; D014867:Water; D062907:Oxaloacetic Acid; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase