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Substrate polarization in enzyme catalysis: QM/MM analysis of the effect of oxaloacetate polarization on acetyl‐CoA enolization in citrate synthase
Author(s) -
van der Kamp Marc W.,
Perruccio Francesca,
Mulholland Adrian J.
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21482
Subject(s) - chemistry , citrate synthase , qm/mm , catalysis , stereochemistry , active site , enzyme , nucleophile , organic chemistry
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 α‐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 α‐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. Proteins 2007. © 2007 Wiley‐Liss, Inc.