4 ��M) was preincubated with ATP (2 mM), ATP-��-S (0.5 mM), ADP (0.5 selleck compound … Effect of temperature on Them1 activity We next measured the temperature dependence of Them1 activity using palmitoyl-CoA (Fig. 5A) and myristoyl-CoA (Fig. 5B) as substrates. For both substrates, values of Km decreased as functions of decreasing temperatures (Fig. 5C), whereas Vmax increased for palmitoyl-CoA and myristoyl-CoA (Fig. 5D). These changes yielded decreases in kcat (Fig. 5E) but much sharper decreases in kcat/Km for Them1 as the temperature was increased from 23��C to 37��C (Fig. 5F). Fig. 5. Temperature dependence of Them1 activity. Saturation curves of V0 for palmitoyl-CoA (A) and myristoyl-CoA (B) were performed at temperatures ranging from 23��C to 45��C. Solid lines indicate fits of the data to the Michaelis-Menten equation.
… Effect of domain structure on thioesterase activity To assess the contributions of the putative functional domains of Them1 on enzyme activity, we used the truncated Them1 constructs displayed in Fig. 1A. The isolated Thio1 domain exhibited thioesterase activity for myristoyl-CoA and palmitoyl-CoA, albeit with higher Km and lower Vmax values compared with Them1 (Fig. 6). By doubling the enzyme concentration, the Km was decreased by 51% and 35% (Fig. 6A), with 20% and 60% increases in Vmax (Fig. 6B), respectively, for the substrates palmitoyl-CoA and myristoyl-CoA. To further characterize this effect, we measured Km and Vmax values for Thio1 compared with Them1 as functions of enzyme concentration using palmitoyl-CoA as the substrate (supplementary Fig.
I). At concentrations of Thio1 less than 0.5 ��M, we did not detect enzyme activity. As the Thio1 concentration was increased from 0.5 ��M, there was a sharp decline of more than 5-fold in values of Km, which leveled off starting at 2 ��M Thio1 (supplementary Fig. IA). By contrast, there was no concentration dependence of Km values for Them1, which were similar to the value observed for the highest Thio1 concentration. Over the same range of protein concentrations, there were modest decreases in Vmax values for Thio1 and little change for Them1 (supplementary Fig. IB). Fig. 6. Influence of Them1 functional domains on acyl-CoA thioesterase activity. Purified recombinant proteins (see Figure 1A) alone or in combination were preincubated at room temperature for 1 h and then assayed for acyl-CoA thioesterase activities at 37��C .
.. The recombinant protein Thio1/2, which contained both thioesterase domains, yielded similar Km and Vmax values as Thio1 but at half the molar concentration of protein (Fig. 6). We next tested the effect of the START domain of Them1. The START domain alone had no thioesterase AV-951 activity. However, when added together with Thio1 at a molar ratio of 1:1, the START domain decreased the Km for palmitoyl-CoA and myristoyl-CoA but increased the Vmax only for myristoyl-CoA.