Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity

Eul Soo Park, jong shik shin

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80% of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K M) rather than the catalytic step (k cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K M and k cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.

Original languageEnglish
Pages (from-to)380-387
Number of pages8
JournalEnzyme and Microbial Technology
Volume49
Issue number4
DOIs
Publication statusPublished - 2011 Sep 10

Fingerprint

Catalyst selectivity
Transaminases
Free energy
Catalytic Domain
Enzymes
Amines
Cats
Substrates
Paracoccus denitrificans
Amination
Ketones
Thermodynamics
Activation energy
Reaction intermediates
Binding energy
Kinetic parameters
Kinetics

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

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abstract = "ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80{\%} of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K M) rather than the catalytic step (k cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K M and k cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.",
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Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity. / Park, Eul Soo; shin, jong shik.

In: Enzyme and Microbial Technology, Vol. 49, No. 4, 10.09.2011, p. 380-387.

Research output: Contribution to journalArticle

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