On the interpretation of quantitative structure-function activity relationship data for lactate oxidase

The native flavin, FMN, has been removed from the L-lactate oxidase of Aero coccus viridans, and the apoprotein reconstituted with 12 FMN derivatives w ith various substituents at the flavin 6- and 8-positions. Impressive linea r relationships are exhibited between the sum of the Hammett sigma(para) an d sigma(ortho) parameters and the redox potentials of the free flavins, and between the redox potentials of the free and enzyme-bound flavins, Rapid r eaction kinetics studies of the reconstituted enzymes with the substrates L -lactate and L-mandelate show an increase in the reduction rate constant wi th increasing redox potential, except that, with lactate, a limiting rate c onstant of approximate to 700 s(-1) is obtained with flavins of high potent ial. Similar breakpoints are found in plots of the rate constants for flavi n N5-sulfite adduct formation and for the reaction of the reduced enzymes w ith molecular oxygen. These results are interpreted in terms of a two-step equilibrium preceding the chemical reaction step, in which the second equil ibrium step provides an upper limit to the rate with which the particular s ubstrate or ligand is positioned with the flavin in the correct fashion for the observed chemical reaction to occur. The relationship of rate constant s for flavin reduction and N5-sulfite adduct formation with flavin redox po tential below the observed breakpoint indicate development of significant n egative charge in the transition states of the reactions, In the case of re duction by substrate, the results are consistent either with a hydride tran sfer mechanism or with the so called "carbanion" mechanism, in which the su bstrate alpha-proton is abstracted by an enzyme base protected from exchang e with solvent. These conclusions are supported by substrate alpha-deuteriu m isotope effects and by solvent viscosity effects on sulfite binding.