Function, structure analysis of human thioesterase superfamily member 2 and determination of 4-chlorobenzoate coenzyme A ligase catalysis mechanism

The work described in this thesis examines structure-function relationships in two enzyme systems: single domain hotdog fold thioesterase THEM2 from human (hTHEM2; Swiss-Prot entry Q9NPJ3) and 4-chlorobenzoate: Coenzyme A ligase (CBAL) from Alcaligenes sp. strain Al3007.;Herein, we report the results of an extensive hTHEM2 substrate screen coupled with the structure determination of hTHEM2 complexed with the inert substrate analog undecan-2-one-CoA (in which O=C-Cl2-S substitutes for O=C-S) and site directed mutagenesis of active site residues. The work reported in this thesis represents the first structure-function based analysis of a human hotdog fold thioesterase.;The mechanism of hydrolysis proposed involves the Asp65 assisted attack of a water molecule at the Gly57/Asn50 polarized thioester C=0 and the Asn50 assisted departure of the thiolate leaving group. Through extensive substrate screening we have evaluated the catalytic prowess of hHTEM2 towards thioesters representing various structural classes. The substrate specificity profile was analyzed within the context of the liganded enzyme to define the structural determinants of substrate recognition. The conservation and variation of these structural recognition elements in bacterial hotdog fold thioesterases of known biochemical function are described. The ultimate conclusion that we reach is that hHTEM2 functions to hydrolyze medium-long chain acyl-CoA thioesters, possibly as part of a system to regulate vectorial fatty acid transport into the cell.;Another enzyme focused on this thesis is the 4-chlorobenzoate: CoA ligase (CBAL) from Alcaligenes sp. strain Al3007. CBAL belongs to the adenylate-forming ligase enzyme superfamily These enzymes share backbone fold and catalytic scaffold, which support the catalysis of thioesterification of a carboxylic acid metabolite. The reaction occurs in two steps: adenylation followed by thioesterification.;The results of steady-state kinetic and transient state kinetic analysis of wild-type CBAL and of a series of site-directed CBAL active site mutants were reported. The major findings are as follows. First, wild-type CBAL is activated by Mg2+ (a 12-75-fold increase in activity is observed depending on assay conditions) and its kinetic mechanism (ping-pong) supports the structure-derived prediction that PPi dissociation must precede the switch from conformation 1 to conformation 2 and therefore CoA binding. Also, transient kinetic analysis of wild-type CBAL identified the rate-limiting step of the catalyzed reaction as one that follows the formation of 4-CB-CoA (viz. CBAL conformational change and/or product dissociation). The single turnover rate of 4-CB and ATP to form 4-CB-AMP and PPi (k = 300 s -1) is not affected by the presence of CoA, and it is ∼3-fold faster than the turnover rate of 4-CB-AMP and CoA to form 4-CB-CoA and AMP (k = 120 s-1). Second, the active site mutants screened via steady-state kinetic analysis were ranked based on the degree of reduction observed in any one of the substrate kcat/ Km values, and those scoring higher than a 50-fold reduction in kcat/Km value were selected for further evaluation via transient state kinetic analysis. The single-turnover time courses, measured for the first partial reaction, and then for the full reaction, were analyzed to define the microscopic rate constants for the adenylation reaction and the thioesterification reaction. On the basis of our findings we propose a catalytic mechanism that centers on a small group of key residues (some of which serve in more than one role) and that includes several residues that function in domain alternation. (Abstract shortened by UMI.)...