Transition-state structural refinement with GRACE and CHARMM: Flexible QM/MM modelling for lactate dehydrogenase

Realistic simulations of chemical reactions require the use not only of methods capable of describing accurately the energy of molecules undergoing bonding changes within a particular chemical environment, but also of methods capable of exploring topographical features of significance on energy hypersurfaces spanning perhaps several thousand degrees of freedom. Hybrid quantum-mechanical/molecular-mechanical techniques show much promise for the first task, but existing computer codes are inadequate for the second. Application of these methods to real chemical problems demands new tools for location and characterisation of saddle-points, intrinsic reaction coordinates, hessians and vibrational frequencies for very large flexible systems. Algorithms capable of performing these tasks have been incorporated in a new software package, GRACE, which provides a non-invasive interface between popular codes for quantum chemistry and molecular dynamics and modelling. Transition structures (TSs) have been refined by this novel procedure, using a combined AM1/CHARMM24/TIP3P potential, involving full gradient relaxation of the positions of 1900–2000 atoms of a solvated enzyme–substrate complex (lactate dehydrogenase/NADH/pyruvate/water). Six different starting structures (arbitrarily selected from a molecular dynamics trajectory for the enzyme–substrate complex) lead to six different TSs. Although the essential features of these TSs are invariant, the relative dispositions of active-site residues differ quite significantly. The transition state for the enzymic reaction would represent an average of the properties of many, nearly degenerate TSs. This insight emerges only as a consequence of the flexible model of the active site employed in this study.