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Human mitochondrial hotdog-fold thioesterase : hTHEM4 and hTHEM5

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Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/12121

Human mitochondrial hotdog-fold thioesterase : hTHEM4 and hTHEM5

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Title: Human mitochondrial hotdog-fold thioesterase : hTHEM4 and hTHEM5
Author: Zhao, Hong
Advisor(s): Dunaway-Mariano, Debra
Committee Member(s): Dunaway-Mariano, Debra
Mariano, Patrick
Bisoffi, Marco
Melancon, Charles
Department: University of New Mexico. Dept. of Chemistry
Subject(s): thioesterase
hotdog-fold
purification
kinetic activity
physiological function
hTHEM4,5
LC Subject(s): Esterases--Structure-activity relationships.
Thiolases--Structure-activity relationships.
Degree Level: Doctoral
Abstract: Hotdog-fold acyl-CoA thioesterases (ACOTs) are found in all three kingdoms of life (Archaea, Bacteria and Eukarya). All the hotdog-fold thioesterases share common catalytic scaffold and function: hydrolysis of acyl-CoA or acyl-ACP (acyl carrier protein). However the biological roles that they perform vary greatly, different substrate specificity and catalytic efficiency assign unique set of biological functions to each of them, which is further defined by tissue distribution, cellular location, protein partners and regulators. The focuses of my doctoral studies, described in this dissertation, are two members of the human hotdog-fold thioesterase superfamily: human THEM4 and human THEM5. The genes encoding hTHEM4 and hTHEM5 are two adjacent brothers in the chromosome 1q21.3. Expression of truncated hTHEM4(∆39) in E. coli produced a homogenous product. In vitro activity analysis proved that hTHEM4 is a high activity, broad substrate range, acyl-CoA thioesterase. In addition to hydrolysis of short and long chain acyl-CoAs (kcat/KM value: 104 to 106 M-1s-1), it also catalyzes the hydrolysis of myristoyl-ACP and palmitoyl-ACP. The X-ray structure of hTHEM4(∆39) bound with an inert fatty acyl-CoA analog revealed that N-terminal domain is two separate helix inserts coupled with a disordered extended loop. In order to understand the relationship between hTHEM4 and Akt1, the effect of purified (fl)hTHEM4 and hTHEM4(∆39) on the phosphorylation (activation) and activity of full-length and truncated Akt1 were tested at GlaxoSmithKline. The results showed that (fl) hTHEM4 has a small but significant direct effect on Akt1 catalytic activity, and also on Akt1 activation at both Thr308 (by PDK1) and Ser473 (by mTORC2). The co-immunoprecipitation experiment was also carried out to investigate the physical association between hTHEM4 and Akt1. Taken together, hTHEM4 and Akt1 are associated with each other, and the effect (inhibition or activation) is concentration-dependent. The full-length hTHEM5 was expressed in HEK 293T cells at a high level compared to that observed for the hTHEM4. The hTHEM5(∆40) X-ray structure depicts essentially the same active site observed for hTHEM4, and no features that we recognize which might explain the difference in expression level in transfected HEK 293T cells. Interestingly, the in vitro kinetic studies carried out with hTHEM5(∆40) showed a similar substrate specificity profile, yet a 100-fold drop in kcat. The cellular location imaging of hTHEM5 in transfected HEK 293T cells using hTHEM5-GFP and (∆32)-hTHEM5-GFP fusions showed specific mitochondrial location of hTHEM5-GFP and a distributive location of the (∆32)-hTHEM5-GFP. In parallel with the biological function study, a structure-function analysis of hTHEM4 and hTHEM5 mechanisms of catalysis and substrate recognition was also carried out. Structure guided site directed mutagenesis and steady-state kinetic analysis of the purified mutants has been done to identify the enzyme residues that contribute to substrate binding and catalysis. Single turnover experiment in H18O was done to define the role of the active carboxylate residue (nucleophilic vs base).
Graduation Date: December 2010
URI: http://hdl.handle.net/1928/12121

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