Figure 6.

ime course of the reaction of V51D GCL with glyoxylate monitored by stopped-flow CD. (A) Formation of 1′,4′-iminoglycolylThDP at pH 7.6 and 6 °C. V51D GCL (3.6 mg/mL, concentration of active centers = 55.6 μM) in 0.1 M KH₂PO₄ (pH 7.6) containing 0.5 mM ThDP, 2.5 mM MgCl₂, 1.0 mM DTT and 10 μM FAD in one syringe was mixed with an equal volume of 2.0 mM glyoxylate in the same buffer in the second syringe. Data were fitted to a double exponential equation (eq 4). (B) The decarboxylation of 1′,4′-iminoglycolyl-ThDP and the formation of TSA-ThDP complex at pH 7.6 and 15 °C. V51D GCL (2.3 mg/mL, concentration of active centers = 35.5 μM) in one syringe was mixed with 2.0 mM glyoxylate in the second syringe at 15 °C. (C) Time-dependent decarboxylation of 1′,4′-iminoglycolylThDP with data from Figure 5 (B) expanded. Data were fitted to a single exponential (eq 5). (D) The formation of TSA-ThDP complex and TSA release by V51D GCL at pH 7.6. The V51D GCL (1.7 mg/mL, concentration of active centers = 26 μM) was pre-incubated with 0.5 mM glyoxylate for 15 min in one syringe, and was then mixed at 6 °C with 16 mM glyoxylate in the second syringe. The data were fitted to a triple exponential (eq 6).