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

Sang Woo Han, Jong Shik Shin

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2 Citations (Scopus)

Abstract

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 (vi) 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/vi 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 determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.

Original languageEnglish
Pages (from-to)S500-S507
JournalJournal of Molecular Catalysis B: Enzymatic
Volume133
DOIs
Publication statusPublished - 2016 Dec 15

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Transaminases
Kinetic parameters
Substrates
Enzymes
Kinetics
Linear regression
Linear Models
Reactive Inhibition
Inhibition (Psychology)
Enzyme kinetics
Amines
Datasets

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Bioengineering
  • Biochemistry
  • Process Chemistry and Technology

Cite this

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title = "A facile method to determine intrinsic kinetic parameters of ω-transaminase displaying substrate inhibition",
abstract = "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 (vi) 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/vi 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 determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.",
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N2 - 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 (vi) 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/vi 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 determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.

AB - 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 (vi) 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/vi 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 determination of four intrinsic kinetic parameters, i.e. turnover number (kcat), substrate inhibition constant (KSI) for A and Michaelis constants (KM) for D and A. To evaluate reliability of the intrinsic parameters, a validity test was taken by comparing experimental and computational results for the maximum point on a concave-down substrate inhibition curve. Once the intrinsic parameters were determined for a substrate pair, intrinsic parameters for other substrates were simply assessed by constituting a new substrate pair with the kinetically characterized substrate and carrying out linear regression with one set of kinetic data. Our method is expected to be applicable to a wide range of bisubstrate enzymes for facile determination of intrinsic kinetic parameters including KSI.

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