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

dc.contributor.advisorWhitman, Christian P.en
dc.creatorRobertson, Brooklyn Ames, 1977-en
dc.date.accessioned2008-08-28T23:39:03Zen
dc.date.accessioned2017-05-11T22:18:12Z
dc.date.available2008-08-28T23:39:03Zen
dc.date.available2017-05-11T22:18:12Z
dc.date.issued2007en
dc.descriptiontexten
dc.description.abstractThe 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.en
dc.description.departmentChemistry and Biochemistryen
dc.description.departmentBiochemistryen
dc.format.mediumelectronicen
dc.identifierb68900405en
dc.identifier.oclc174269392en
dc.identifier.urihttp://hdl.handle.net/2152/3288en
dc.language.isoengen
dc.rightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.en
dc.subject.lcshTautomerasesen
dc.titleEvolution 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 glutamicumen
dc.title.alternativePresteady state kinetic analysis of cis-3-chloroacrylic acid dehalogenase and an inhibition study of its homologue, cg10062, in corynebacterium glutamicumen
dc.type.genreThesisen

Files