Modelling flavin and substrate substituent effects on the activation barrier and rate of oxygen transfer by p-hydroxybenzoate hydroxylase

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 der ivatives catalysed by wild-type para-hydroxybenzoate hydroxylase (PHBH) as well as for the hydroxylation of the native substrate (p-hydroxybenzoate) b y PHBH reconstituted with a series of 8-substituted flavins. The following relative parameters have been calculated and tested: (a) energy barriers fr om combined quantum mechanical/molecular mechanical (QM/ MM) (AM1/CHARMM) r eaction pathway calculations, (b) gasphase 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 gasphase approaches y ielded good correlations, as long as similarly charged species are involved , The QM/MM approach resulted in a good correlation, even including differe ntly charged species. This indicates that the QM/MM model accounts quite we ll for the solvation effects of the active site surroundings, which vary fo r differently charged species. The correlations obtained demonstrate quanti tative structure activity relationships for an enzyme-catalysed reaction in cluding, for the first time, substitutions on both substrate and cofactor, (C) 2000 Federation of European Biochemical Societies. Published by Elsevie r Science B.V. All rights reserved.