Indicative of its role in oligomerization, acetyl-CoA inhibits cold inactivation of Acot12 (24) and facilitates reactivation of the enzyme upon warming (17). Our finding especially that acetyl-CoA promotes Acot12 tetramer formation is in accordance with these observations. Although temperature per se did not influence oligomerization of Them1, its dimerization was effected by the presence of a fatty acyl-CoA. Collectively, these findings demonstrate that the substrates of Acot12 and Them1 mediate oligomerization. ATP promotes tetramerization of Acot12 (25) and its activation (18). Here we showed that it also induced dimerization and increased activity for Them1. Like Acot12 (18), ATP hydrolysis was not required for the activation of Them1, as evidenced by similar effects of ATP and ATP-��-S on oligomerization and enzymatic activity.

ADP induced Them1 to dimerize yet decreased its activity. This is in keeping with the observation that ADP promotes tetramerization of Acot12 while at the same inactivating the enzyme (26). The effects of ATP and ADP on Acot12 were attributed to the potential presence of a nucleotide binding site in proximity to the enzyme active site (26). Because nucleotide effects on Acot12 activity were consistent with simple competitive displacements, it was concluded that ATP and ADP more likely induce conformational changes that lead to different catalytic activities and substrate affinities. Consistent with this possibility for Them1, Lineweaver-Burk plots were indicative of a mixed mechanism of inhibition.

The mechanism by which CoASH inhibits Acot12 (18) and Them1 is not known but may be due to the presence of ADP within its molecular structure. With the exception of Acot12, medium- and long-chain acyl-CoAs are generally the preferred substrates of type II Acots (1), an observation that extends to the more recently characterized Them4 (22) and Them5/Acot15 (23). Although previously suggested to hydrolyze medium- and long-chain fatty acyl-CoAs (9), in the current study Them1 cleaved a range of substrates, albeit demonstrating a relative preference for medium- to long-chain fatty acyl-CoAs. The steady-state kinetic constants for fatty acyl-CoA hydrolysis for Them1 fall in similar ranges as those of Them2 (8), Them4 (22), and Them5 (23).

When characterized using a short- and a long-chain fatty acyl-CoA substrate, THEM1a and THEM1b yielded similar kinetic constants as Them1, suggesting that the additional Carfilzomib 13 aa at the C terminus of THEM1a (9) serves a function separate from the enzymatic activity of the protein. The intracellular concentrations of fatty acyl-CoAs remain an important unresolved issue in understanding the biologic functions of Acots. Total intracellular concentrations of long-chain fatty acyl-CoAs range from 5 ��M in neutrophils to 164 ��M in liver (27), with estimates of 30�C90 ��M in cytosol, 0.2�C3.1 mM in mitochondria, and 0.4 mM in peroxisomes (2).