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.