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Characterization of the meta-cleavage pathway enzymes, 4-Hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating), from Pseudomonas sp. strain CF600

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Characterization of the meta-cleavage pathway enzymes, 4-Hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating), from Pseudomonas sp. strain CF600

Monastiriakos, Stavroula (1998) Characterization of the meta-cleavage pathway enzymes, 4-Hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating), from Pseudomonas sp. strain CF600. Masters thesis, Concordia University.

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Abstract

The work presented in this thesis focuses on three aspects of the aldolase-dehydrogenase. First, DmpG was overexpressed and purified in order to address the question of whether this polypeptide alone possesses 4-hydroxy-2-ketovalerate aldolase activity. While previous work had indicated that DmpF alone can catalyze dehydrogenase activity, thus far no aldolase activity has been detected in preparations of DmpG. Second, steady-state kinetics studies of the individual and coupled reactions were performed in order to address the question of coupling. The data obtained are consistent with channeling of the aldolase product, acetaldehyde, to the dehydrogenase active site. Furthermore, kinetics measurements indicate either a partially or fully ordered terreactant mechanism for the dehydrogenase. Finally, chemical modification of the enzyme by iodoacetate provided evidence for the involvement of a reactive cysteine residue in dehydrogenase, but not aldolase, activity. This result is consistent with postulated mechanisms of other aldehyde dehydrogenases, and will allow creation of a variant that can be used to study the reactions of the aldoase separately from the dehydrogenase partner.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Monastiriakos, Stavroula
Pagination:xii, 96 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (M.Sc.)
Program:Chemistry and Biochemistry
Date:1998
Thesis Supervisor(s):Powlowski, Justin B
ID Code:572
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:12
Last Modified:08 Dec 2010 10:15
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