Molecular Mechanism of Methanol Inhibition in CALB-Catalyzed Alcoholysis: Analyzing Molecular Dynamics Simulations by a Markov State Model

• Main Authors: Henrique F. Carvalho (11408021), Valerio Ferrario (651292), Jürgen Pleiss (1723024)
• Format: Other Non-Article Part of Journal/Newspaper
• Language: unknown
• Published: 2021
• Subjects: Biophysics; Biochemistry; Genetics; Molecular Biology; Pharmacology; Biotechnology; Inorganic Chemistry; Biological Sciences not elsewhere classified; Chemical Sciences not elsewhere classified; widely used enzymes; markov state models; fatty acid esters; experimental activity profile; substrate binding kinetics; substrate access channel; structural analysis uncovered; unbiased molecular dynamics; kinetic bottleneck results; derive kinetic information; productive methanol binding; methanol patches close; beyond 300 mm; realistic substrate concentrations; unbiased md simulations; 300 mm; msm analysis; molecular mechanism; molecular basis; 50 mm; small alcohols; msms ); modeled fluxes; many lipases; high concentrations; helix α10; gradual formation; good agreement; findings demonstrate; catalyze hydrolysis; active site; Antarc*; Antarctica
• Online Access: https://doi.org/10.1021/acs.jctc.1c00559.s001

Lipases are widely used enzymes that catalyze hydrolysis and alcoholysis of fatty acid esters. At high concentrations of small alcohols such as methanol or ethanol, many lipases are inhibited by the substrate. The molecular basis of the inhibition of Candida antarctica lipase B (CALB) by methanol was investigated by unbiased molecular dynamics (MD) simulations, and the substrate binding kinetics was analyzed by Markov state models (MSMs). The modeled fluxes of productive methanol binding at concentrations between 50 mM and 5.5 M were in good agreement with the experimental activity profile of CALB, with a peak at 300 mM. The kinetic and structural analysis uncovered the molecular basis of CALB inhibition. Beyond 300 mM, the kinetic bottleneck results from crowding of methanol in the substrate access channel, which is caused by the gradual formation of methanol patches close to Leu140 (helix α5), Leu278, and Ile285 (helix α10) at a distance of 4–5 Å from the active site. Our findings demonstrate the usefulness of unbiased MD simulations to study enzyme–substrate interactions at realistic substrate concentrations and the feasibility of scale-bridging by an MSM analysis to derive kinetic information.