A facile method to determine intrinsic kinetic parameters of ω-transaminase displaying substrate inhibition

Publication date: Available online 8 May 2017 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Sang-Woo Han, Jong-Shik Shin It is usually time-consuming to determine intrinsic kinetic parameters of bisubstrate enzymes, especially when experimental kinetic data deviate from a linear Lineweaver-Burk plot due to complex inhibition patterns. A typical example is ω-transaminase (ω-TA) which is an industrially important enzyme for asymmetric synthesis of chiral amines. ω-TA catalyzes transfer of an amino group between a donor (D) and an acceptor (A) via a ping-pong bi-bi mechanism and often displays substrate inhibitions by reactive amino acceptors, which leads one to prefer to determine apparent kinetic parameters rather than intrinsic ones despite limited applicability for precise understanding of enzyme properties. Here, we developed a new method to determine intrinsic kinetic parameters of ω-TA by double-reciprocal analysis using only two sets of kinetic data. First, linear regression of 1/initial rate (v i) against 1/[A] was carried out with one set of kinetic data measured at a fixed [D] while [A] lay far below the concentration range under the influence of substrate inhibition. Second, another linear regression of 1/[D] vs 1/v i was conducted with one set of kinetic data obtained at a fixed [A] within a substantial substrate inhibition range. The resulting four equations obtained from the y-intercepts and slopes of the two regression lines were used for ...

Source: Journal of Molecular Catalysis B: Enzymatic - May 9, 2017 Category: Biochemistry Source Type: research