Inactivation of wild-type GlmU acetyltransferase by thiol-specific reagents. (A) Wild-type GlmU was stripped of β-mercaptoethanol and incubated at 37°C in 20 mM potassium phosphate buffer in the absence (•) or presence of 14 mM β-mercaptoethanol (◊), 1.6 mM acetyl-CoA (⧫), 4 mM GlcN-1-P (○), 2.7 mM GlcNAc-1-P (□), or 1 mM UTP (■). At the indicated times, aliquots (1.8 ng of protein) were removed and the residual acetyltransferase activity was measured. The activity at t = 0 was 30% lower in the presence of GlcNAc-1-P because this reaction product inhibits the enzyme (19). (B) Wild-type GlmU was stripped of β-mercaptoethanol and incubated at 37°C in the absence (•) or presence of various thiol-specific reagents: NEM at 50 μM (■), DTNB at 5 μM (○), and NTCB at 50 μM (⧫). Samples (1.8 ng of protein) were removed at the indicated times, and the residual acetyltransferase activity was measured. Curves obtained with iodoacetamide at 100 μM and pHMB at 2.5 μM could be superimposed on that obtained with NEM at 50 μM. □, enzyme incubated with 1.6 mM acetyl-CoA for 30 s prior to addition of NEM at a final concentration of 50 μM. (C) Wild-type GlmU was stripped of β-mercaptoethanol and incubated at 37°C in 20 mM potassium phosphate buffer containing 5 μM DTNB. At t = 1.5 min (arrow), the mixture was divided in two parts, and β-mercaptoethanol was added to one at a final concentration of 14 mM. Samples (1.8 ng of protein) were removed at different times, and the residual acetyltransferase activity was measured. Symbols: •, no addition; ■, addition of β-mercaptoethanol at t = 1.5 min. All experiments were performed at least in duplicate, and standard deviations were less than 10%.