The role of molecular interactions on Michaelis constants of α-chymotrypsin catalyzed peptide hydrolyses. (September 2020)

• Record Type: Journal Article
• Authors: Knierbein, Michael; Held, Christoph; Sadowski, Gabriele
• Is Part Of: Journal of chemical thermodynamics. Volume 148(2020)
• Publication Date: 2020-09
• Subjects: Enzyme kinetics -- Modeling -- PC-SAFT -- Thermodynamic activities -- Solubility; Thermodynamics -- Periodicals; Thermochemistry -- Periodicals; Thermodynamique -- Périodiques; Thermochimie -- Périodiques; Thermochemistry; Thermodynamics; Periodicals; 541.369
• DOI: 10.1016/j.jct.2020.106142
• Languages: English
• ISSNs: 0021-9614

Abstract

Highlights: Solubility of L -phenylalanine- p -nitroanilide was measured in different solvents. Pressure decreases Michaelis constant KM for hydrolyses of HPNA and SPNA. TMAO and DMSO decrease KM of HPNA while they increase KM of SPNA. Co-solvent and pressure effects on KM were predicted correctly with PC-SAFT. Substrate/co-solvent molecular interactions (activity coefficient) determine KM. Abstract: In this work, the effects of co-solvent and pressure on Michaelis constants at ambient temperature were analyzed for the enzymatic peptide hydrolyses of L -phenylalanine- p -nitroanilide (HPNA) and of N-succinyl- L -phenylalanine- p -nitroanilide (SPNA). These two substrates resemble each other in their molecular structure. That is, at the position of SPNA's succinyl-group (S), HPNA possesses a hydrogen atom (H). Two co-solvents were considered: trimethylamine N-oxide and dimethyl sulfoxide. The thermodynamic model Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) was used to predict the activity of HPNA and SPNA under different reaction conditions regarding solvent composition and pressure. The PC-SAFT parameters (pure-component parameters and one binary parameter between substrate and solvent) were fitted to solubility data of HPNA in different solvents (water, ethanol, ethyl acetate, dimethyl sulfoxide), which were measured in this work at 30 °C and 1 bar. The resulting PC-SAFT predicted Michaelis constants were validated by experimental literature data. Results show that pressure decreased the Michaelis constants of both reactions, HPNA hydrolysis and SPNA hydrolysis. In spite of that, co-solvent effects on the Michaelis constants were predicted to be contrary for the two hydrolysis reactions. For the hydrolysis of HPNA, the co-solvents under investigation decreased the Michaelis constant while the co-solvents increased the Michaelis constant for the hydrolysis of SPNA. These PC-SAFT predictions were in qualitative agreement with the experimental literature data. This shows that molecular interactions are the key to understand the effects of co-solvents on Michaelis constants for the considered reactions. Applying the thermodynamic model PC-SAFT allowed predicting the observed combined effects of co-solvent and pressure on enzymatic reaction kinetics, which opens the door for solvent design of enzymatic reactions in the future.