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Evolution and divergence in the tautomerase superfamily: a presteady state kinetic analysis of cis-3-chloroacrylic acid dehalogenase and an inhibition study of its homologue, cg10062, in corynebacterium glutamicum / Presteady state kinetic analysis of cis-3-chloroacrylic acid dehalogenase and an inhibition study of its homologue, cg10062, in corynebacterium glutamicumRobertson, Brooklyn Ames, 1977- 28 August 2008 (has links)
The tautomerase superfamily is a group of structurally homologous proteins characterized by a [beta-alpha-beta] building block and a catalytic amino-terminal proline (Pro-1). The isomer specific hydrolytic dehalogenases, cis- and trans-3-chloroacrylic acid dehalogenase, (cis-CaaD and CaaD, respectively) are two superfamily members found in bacterial pathways for the catabolism of the nematocide 1,3-dichloropropene. The enzyme-catalyzed addition of water produces two products, malonate semialdehyde and a halide ion. Although the enzymes share a common catalytic tetrad, there are two notable differences: two additional residues have been implicated in the cis-CaaD mechanism and mutagenesis analysis of the core catalytic residues suggests varying degrees of importance. As part of an effort to understand the origin of these differences, a pre steady state kinetic analysis of cis-CaaD was carried out. For the analysis, an ionexchange method was developed for bromide quantification. The analysis produced a five-step kinetic model in which substrate binding is followed by a conformational change. Halide ion is released first in the rate limiting step followed by the release of malonate semialdehyde. The stage is now set for a similar analysis of CaaD and the cisCaaD and CaaD mutants. In the second part of the dissertation, (R)- and (S)-oxirane-2-carboxylate were determined to be active-site-directed irreversible inhibitors of the cisCaaD homologue designated Cg10062 and found in Corynebacterium glutamicum. Kinetic analysis indicates that the (R)-enantiomer binds more tightly and is the more potent inhibitor. Pro-1 is the sole site of modification by the (R)- and the (S)-enantiomer. The results are similar to those found for the irreversible inactivation of cis-CaaD by (R)-oxirane-2-carboxylate with an important distinction: the alkylation of cis-CaaD is stereospecific. Cg10062 exhibits a relaxed substrate specificity processing both the cis- and trans-3-chloroacrylic acid. Delineation of the factors responsible for the stereoselective inactivation would provide a more complete picture of the substrate specificity determinants for cis-CaaD and CaaD. / text
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Evolution and divergence in the tautomerase superfamily a presteady state kinetic analysis of cis-3-chloroacrylic acid dehalogenase and an inhibition study of its homologue, cg10062, in corynebacterium glutamicum /Robertson, Brooklyn Ames, January 1900 (has links)
Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.
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