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 met hods capable of describing accurately the energy of molecules undergoing bo nding changes within a particular chemical environment, but also of methods capable of exploring topographical features of significance on energy hype rsurfaces spanning perhaps several thousand degrees of freedom. Hybrid quan tum-mechanical/molecular-mechanical techniques show much promise for the fi rst task, but existing computer codes are inadequate for the second. Applic ation of these methods to real chemical problems demands new tools for loca tion and characterisation of saddle-points, intrinsic reaction coordinates, hessians and vibrational frequencies for very large flexible systems. Algo rithms capable of performing these tasks have been incorporated in a new so ftware package, GRACE, which provides a non-invasive interface between popu lar codes for quantum chemistry and molecular dynamics and modelling. Trans ition structures (TSs) have been refined by this novel procedure, using a c ombined AM1/CHARMM24/TIP3P potential, involving full gradient relaxation of the positions of 1900-2000 atoms of a solvated enzyme-substrate complex (l actate dehydrogenase/NADH/pyruvate/water). Six different starting structure s (arbitrarily selected from a molecular dynamics trajectory for the enzyme -substrate complex) lead to six different TSs. Although the essential featu res of these TSs are invariant, the relative dispositions of active-site re sidues differ quite significantly. The transition state for the enzymic rea ction would represent an average of the properties of many, nearly degenera te TSs. This insight emerges only as a consequence of the flexible model of the active site employed in this study.