Functional Characterization of Corynebacterium glutamicum Mycothiol S-Conjugate Amidase
Figure 3
Catalytic activity of C. glutamicum Mca.
A. GlcN standard curve. Solutions of GlcN (0–100 µM) in buffer (50 mM HEPES, 50 mM NaCl, and 1 mM TCEP, pH 7.5) were diluted with borate (0.75 M, pH 9.0) and mixed with FSA (2.3 mM), followed by measurement of the resulting fluorescence. The observed increase in fluorescence was drawn against glucosamine concentration to generate the standard curve. A linear equation was fitted to data. B. Mca-catalyzed reaction. GlcNAc (2 mM) was pre-incubated at 30°C in assay buffer (50 mM HEPES, 50 mM NaCl, and 1 mM TCEP, pH 7.5) and the reaction was started by the addition of Mca (6.4 µM). At different time points, aliquots of reaction mixture were terminated by 5% trichloroacetic acid, diluted with borate (0.75 M, pH 9.0) and mixed with FSA (2.3 mM), followed by measurement of the resulting fluorescence. The glucosamine standard curve (A) was used to transform the observed rate of the reaction into µ min−1.