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Functional and regulatory mechanisms in alpha-isopropylmalate synthases

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dc.contributorBusenlehner, Laura S.
dc.contributorWoski, Stephen A.
dc.contributorStreet, Shane C.
dc.contributorMarcus, Stevan
dc.contributor.advisorFrantom, Patrick A.
dc.contributor.authorCasey, Ashley Kay
dc.contributor.otherUniversity of Alabama Tuscaloosa
dc.date.accessioned2017-03-01T17:21:51Z
dc.date.available2017-03-01T17:21:51Z
dc.date.issued2014
dc.descriptionElectronic Thesis or Dissertationen_US
dc.description.abstractThe allosteric regulation of a protein is where the binding of a molecule at a distal site affects the physical and chemical properties at the binding site. A model system for studying allosteric mechanism is isopropylmalate synthase isolated from Mycobacterium tuberculosis (MtIPMS). MtIPMS catalyzes a Claisen-like condensation between acetyl-CoA and ketoisovalerate to form the products isopropylmalate and CoA, which is the first committed step in the biosynthesis of L-leucine. L-Leucine acts as a slow-onset feedback inhibitor binding 50 Å from the active site in the regulatory domain. Structural studies of MtIPMS indicate that a flexible loop becomes more ordered upon L-leucine binding. Alternate amino acid inhibitors and site-directed mutagenesis results indicate this flexible loop plays a role in the slow-onset mechanism of MtIPMS. Kinetically, L-leucine acts as a V-type inhibitor, lowering V_max for the reaction while K_m values remain relatively unchanged. A decrease in V_max could be caused by a decrease in the rate of a chemical step or product release. Results from rapid-reaction kinetics and kinetic isotope effects indicate that the rate-limiting step shifts from product release to hydrolysis upon the binding of L-leucine. Hydrogen/deuterium exchange experiments indicated that upon L-leucine binding a helix in the active site cavity undergoes a conformational change suggesting that it could be involved in the allosteric mechanism of MtIPMS. The results from site-directed mutagenesis studies indicate that this active site helix is not involved in the allosteric mechanism of MtIPMS. Isopropylmalate synthase isolated from Francisella novicida (FnIPMS) shares a sequences identity of 26% with MtIPMS over 526 residues. This is the first report of a monomeric IPMS to date. The kinetic parameters of FnIPMS are comparable to that of MtIPMS. However, the K_i value is approximately 150-fold higher than that of MtIPMS. Kinetic isotope effects also indicate that hydrolysis is the rate-limiting step in the presence of L-leucine.en_US
dc.format.extent165 p.
dc.format.mediumelectronic
dc.format.mimetypeapplication/pdf
dc.identifier.otheru0015_0000001_0001780
dc.identifier.otherCasey_alatus_0004D_12158
dc.identifier.urihttps://ir.ua.edu/handle/123456789/2226
dc.languageEnglish
dc.language.isoen_US
dc.publisherUniversity of Alabama Libraries
dc.relation.hasversionborn digital
dc.relation.ispartofThe University of Alabama Electronic Theses and Dissertations
dc.relation.ispartofThe University of Alabama Libraries Digital Collections
dc.rightsAll rights reserved by the author unless otherwise indicated.en_US
dc.subjectChemistry
dc.subjectBiochemistry
dc.titleFunctional and regulatory mechanisms in alpha-isopropylmalate synthasesen_US
dc.typethesis
dc.typetext
etdms.degree.departmentUniversity of Alabama. Department of Chemistry
etdms.degree.disciplineChemistry
etdms.degree.grantorThe University of Alabama
etdms.degree.leveldoctoral
etdms.degree.namePh.D.

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