The simulation of enzymatic reactions, using computer models, is becoming a powerful tool in the most fundamental challenge in biochemistry: to relate the catalytic activity of enzymes to their structure. In the present study, various computed parameters were correlated with the natural logarithm of experimental rate constants for the hydroxylation of various substrate derivatives catalysed by wild-type para-hydroxybenzoate hydroxylase (PHBH) as well as for the hydroxylation of the native substrate (p-hydroxybenzoate) by PHBH reconstituted with a series of 8-substituted flavins. The following relative parameters have been calculated and tested: (a) energy barriers from combined quantum mechanical/molecular mechanical (QM/MM) (AM1/CHARMM) reaction pathway calculations, (b) gas-phase reaction enthalpies (AM1) and (c) differences between the HOMO and LUMO energies of the isolated substrate and cofactor molecules (AM1 and B3LYP/6-31+G(d)). The gas-phase approaches yielded good correlations, as long as similarly charged species are involved. The QM/MM approach resulted in a good correlation, even including differently charged species. This indicates that the QM/MM model accounts quite well for the solvation effects of the active site surroundings, which vary for differently charged species. The correlations obtained demonstrate quantitative structure activity relationships for an enzyme-catalysed reaction including, for the first time, substitutions on both substrate and cofactor.
