Reliable kinetic parameters of enzymes are of paramount importance for a precise understanding of catalytic performance, which is essential for enzyme engineering and process optimization. Here, we developed a simple and convenient method to determine intrinsic kinetic parameters of R-selective ω-transaminases (ω-TAs) with a minimal set of kinetic data. Using (R)-α-methylbenzylamine ((R)-α-MBA) and pyruvate as a substrate pair, two R-selective ω-TAs from Arthrobacter sp. and Aspergillus fumigatus were subjected to kinetic measurements. In contrast to S-selective ω-TAs, both R-selective ω-TAs were observed to be devoid of substrate inhibition by pyruvate. Double reciprocal plot analysis was carried out with two sets of kinetic data obtained at varying concentrations of (R)-α-MBA under a fixed concentration of pyruvate and vice versa, leading to the determination of three intrinsic kinetic parameters, i.e., one kcat and two KM values, using three regression constants. The validity of the kinetic parameters was verified by a self-consistency test using a regression constant left out in the kinetic parameter determination, showing that deviations of calculated regression constants from the experimental ones were less than 15%. Because the kinetic parameters for (R)-α-MBA and pyruvate are not apparent but intrinsic, a cosubstrate substitution method enabled rapid determination of intrinsic parameters for a new substrate pair using just one set of kinetic data. Eventually, computational modeling of kinetic resolution of rac-α-MBA was carried out and showed a good agreement with experimental reaction progresses.
Bibliographical noteFunding Information:
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2019R1F1A1062845). Dr. S.-W. Han was financially supported by the Initiative for Biological Function & Systems under the BK21 PLUS program of the Korean Ministry of Education.
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All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology
- Molecular Biology