4.4 Article

Kinetic Analysis of R-Selective ω-Transaminases for Determination of Intrinsic Kinetic Parameters and Computational Modeling of Kinetic Resolution of Chiral Amine

Journal

APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 191, Issue 1, Pages 92-103

Publisher

SPRINGER
DOI: 10.1007/s12010-020-03240-x

Keywords

Kinetic modeling; Intrinsic parameter; omega-Transaminase; Chiral amine; Computational modeling

Funding

  1. National Research Foundation of Korea (NRF) - Korean government (MSIT) [2019R1F1A1062845]
  2. Initiative for Biological Function & Systems under the BK21 PLUS program of the Korean Ministry of Education
  3. National Research Foundation of Korea [2019R1F1A1062845] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)

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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 omega-transaminases (omega-TAs) with a minimal set of kinetic data. Using (R)-alpha-methylbenzylamine ((R)-alpha-MBA) and pyruvate as a substrate pair, two R-selective omega-TAs from Arthrobacter sp. and Aspergillus fumigatus were subjected to kinetic measurements. In contrast to S-selective omega-TAs, both R-selective omega-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)-alpha-MBA under a fixed concentration of pyruvate and vice versa, leading to the determination of three intrinsic kinetic parameters, i.e., one k(cat) and two K-M 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)-alpha-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-alpha-MBA was carried out and showed a good agreement with experimental reaction progresses.

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