A phenylalanine dynamic switch controls the interfacial activation of Rhizopus chinensis lipase

The catalytic mechanism of most lipases involves a step called interfacial activation which significantly increases lipases activity beyond the critical micellar concentration (CMC) of substrate. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of lipase interfacial activation beyond the CMC. Molecular dynamic (MD) simulations indicated the open- and closed-lid transitions and revealed that Phe113 was the critical site for RCL activation by its dynamic flipping. Such putative switch affecting interfacial activation has not been reported in lipase so far. The function of Phe113 was subsequently verified by mutation experiments. The F113Wmutant increases the lipase catalytic efficiency (1.9 s(-1).mu M-1) to 280% at the optimum temperature (40 degrees C) and pH 8.5 with the addition of 0.12 mu g protein in the 200 mu L reaction system. MD simulations indicated that the fast flipping rate from the closed to the open state, the high open state proportion, and the exposure of the catalytic triad are the main reasons for the lipase activation. The mutual corroboration of simulations and site-directed mutagenesis results revealed the vital role of Phe113 in the lipase activation. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

The study revealed the crucial role of Phe113 in the interfacial activation of Rhizopus chinensis lipase, showing that it plays a dynamic flipping role in activation. Mutation experiments confirmed the function of Phe113, with the fast flipping rate, high open state proportion, and exposure of the catalytic triad identified as the main reasons for lipase activation. The mutual corroboration of simulations and site-directed mutagenesis results highlighted the critical role of Phe113 in lipase activation.