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dc.contributor.advisor San, Ka-Yiu
dc.creatorWiesenborn, Dennis Paul
dc.date.accessioned 2009-06-04T00:01:48Z
dc.date.available 2009-06-04T00:01:48Z
dc.date.issued 1989
dc.identifier.urihttps://hdl.handle.net/1911/16310
dc.description.abstract Three key enzymes of the Clostridium acetobutylicum ATCC 824 metabolism, namely thiolase, phosphotransbutyrylase (PTB), and CoA transferase, were purified to homogeneity. Each of these enzymes catalyzes the first reaction after a branch-point in the fermentative pathway, and may therefore be an important point in the regulation of the fermentation. A number of physical and kinetic properties, namely native and subunit molecular weight, K$\sb{\rm m}$ values, kinetic binding mechanism, optimum pH, substrate specificity, and effect of cofactors and metabolites on enzyme activity were determined with the objective of identifying important regulatory factors for each enzyme. Thiolase is inhibited by micromolar levels of CoASH in the condensation direction, and this may be the most important factor in modulating the net rate of condensation of acetyl CoA. The enzyme is relatively insensitive to change in pH within the physiologic range. Studies of thiolase specific activity under various types of continuous fermentations show that regulation of this enzyme at both the genetic and enzyme level is important. PTB is sensitive to pH change in the butyryl phosphate-forming direction, within the physiologic range of pH 5.5 to 7; relative activity decreases with decreasing pH to about 5% at pH 6. This indicates that change in internal pH may be one important factor in the regulation of the enzyme. The enzyme can use a number of substrates in addition to butyryl CoA, but has the highest relative activity with butyryl CoA, isovaleryl CoA and valeryl CoA. CoA transferase is very unstable in vitro unless high concentrations of both glycerol (20%) and ammonium sulfate (0.5 M) are present; virtually all activity is lost within an hour without either stabilizer. The K$\sb{\rm m}$ values for butyrate and acetate of 660 mM and 1200 mM, respectively, are high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase is able to activate propionate, valerate, isobutyrate, and crotonate. The enzyme activity is relatively independent of pH, but is inhibited by physiologic levels of acetone and butanol, suggesting a feedback inhibition mechanism.
dc.format.extent 167 p.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subjectChemical engineering
Biochemistry
dc.title Thiolase, phosphotransbutyrylase, and coenzyme-A transferase and their role in product formation in {\it Clostridium acetobutylicum\/} ATCC 824
dc.type.genre Thesis
dc.type.material Text
thesis.degree.department Chemistry
thesis.degree.discipline Natural Sciences
thesis.degree.grantor Rice University
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy
dc.identifier.citation Wiesenborn, Dennis Paul. "Thiolase, phosphotransbutyrylase, and coenzyme-A transferase and their role in product formation in {\it Clostridium acetobutylicum\/} ATCC 824." (1989) Diss., Rice University. https://hdl.handle.net/1911/16310.


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