Kinetics of the exchange reaction catalyzed by 2-amino-3- ketobutyrate CoA ligase

Abstract

2-Amino-3-ketobutyrate CoA ligase (KBL) of Escherichia coli is a member of the α-oxoamine synthase family; it catalyzes the condensation reaction between glycine and acetyl CoA to yield 2-amino-3-ketobutyrate. We have previously shown that KBL catalyzes the exchange of pro-R hydrogen of glycine with protons in the medium; however, the kinetics of this reaction has never been determined. In this study, we calculated the kinetic parameters of this exchange reaction by using different concentrations of [2RS- ³H₂: 2-¹⁴C] glycine. The rate of the exchange reaction was determined by measuring the ³H/¹⁴C ratio in recovered [2S- ³H: 2-¹⁴C]glycine. The Lineweaver-Burk plot showed that K_m and k_cat of this reaction were 3.8 ×10^-3 M and 0.22 S^-1, respectively. On the other hand, K_m and k_cat values of the overall KBL-mediated catalysis were correspondingly 1.23 × 10^-2 M and 1.19 S^-1. Thus, the rate of the exchange reaction was almost five times lower than that of overall KBL catalysis.

Keywords

enzyme / 2-amino-3-ketobutyrate CoA ligase / kinetics / exchange reaction

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Acknowledgments

I am grateful to the Higher Education Commission (HEC) of Pakistan for grants that enabled establishment of the Bioinformatics research laboratory at COMSATS, Sahiwal.

This study does not contain any experiment on human and animals by the author.

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg