Kinetic studies on the lignin peroxidase catalyzed oxidation of veratryl alcohol by H2O2 in AOT/isooctane/toluene/water reverse micelles

The steady state kinetics of the lignin peroxidase (LiP) catalyzed oxidation of veratryl alcohol (VA) by H2O2 in a sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane/toluene/water reverse micellar medium was studied and a comparison with the corresponding aqueous medium was made to understand the effect of the reverse micellar medium on the catalytic mechanism and kinetic parameters. Results indicated that the model reaction in the AOT reverse micelle followed the ping-pong mechanism with true k(cat), K-m,(VA) and K-m,K-H2O2 being 59.6 min-1, 13.9 mmol center dot L-1 and 94.8 mu mol center dot L-1, respectively; inhibition of high level of H2O2 on UP followed the reversible competitive pattern with K-i being 0.140 mmol center dot L-1. The reaction mechanism and inhibition pattern in the AOT reverse micellar medium were the same as those in bulk aqueous medium, but the kinetic parameters except K-m,K-H2O2 were greatly different in the two media. The kcat and Ki values in the reverse micelle were approximately 2 and 20 times smaller than the corresponding values in the aqueous solution, but the Michaelis constant of VA was approximately 100 times greater than that in the aqueous solution. The above mentioned differences in the kinetic parameters were caused by the microbeterogeneity and the interface of the AOT reverse micelle, which resulted in the partitioning of VA and H2O2, and by the changes of the conformation of UP and the reactivity of the substrates.