The Mechanisms of Human Hotdog-fold Thioesterase 2 (hTHEM2) Substrate Recognition and Catalysis Illuminated by a Structure and Function Based Analysis

The focus of this paper is the hotdog-fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry). In an earlier communication (Cheng, Z., Song, F., Shan, X., Wei, Z., Wang, Y., Dunaway-Mariano, D., and Gong, W. (2006) Crystal structure of human thioesterase superfamily member 2, Biochem. Biophys. Res. Comintin. 349, 172-177) we reported the apo crystal structure of hTHEM2. Herein, we report the results of an extensive hTHEM2 substrate screen, the structure determination of hTHEM2 complexed with the inert substrate analogue undecan-2-one-CoA (in which O=C-CH2-S substitutes for O=C-S) and the kinetic analysis of active site mutants. The work described in this paper represents the first reported structure-function based analysis of a human hotdog-fold thioesterase. The catalytic mechanism proposed involves the Asp65/Scr83 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester C=O and the Asn50 assisted departure of the thiolate leaving group. Thioesterase activity was observed with acyl-CoAs but not with the human acyl-ACP or with an acyl-Cys peptide. The medium-to-long-chain fatty acyl-CoAs displayed the smallest K-m values. The substrate specificity profile was analyzed within the context or the liganded enzyme to define the structural determinants of substrate recognition. Based on the results of this structure-function analysis we hypothesize that the physiological role of hTHEM2 involves catalysis of the hydrolysis of cytosolic medium-to-long-chain acyl-CoA thioesters.