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dc.contributor.advisorChairperson, Graduate Committee: John W. Peters.en
dc.contributor.authorPandey, Arti Sharma.en
dc.date.accessioned2013-06-25T18:37:42Z
dc.date.available2013-06-25T18:37:42Z
dc.date.issued2007en
dc.identifier.urihttps://scholarworks.montana.edu/xmlui/handle/1/2005
dc.description.abstractX-ray crystallography was employed to probe the mechanism of the enzyme 2- ketopropyl coenzymeM oxidoreductase / carboxylase (2-KPCC). We were able to determine the enzyme structure in various catalytically relevant states, providing insights into substrate binding, intermediate stabilization, product formation and release. Structures of 2-KPCC were obtained with the substrate 2-ketopropyl coenzyme M (KCoM), product acetoacetate, 6-oxoheptanoic acid (OHA), 2-oxopropyl phosphonate (OPP), NADP+ and coenzymeM (CoM), the oxidized and reduced states. The binding sites for these ligands in relation to one another have led to important sights into the mechanism. CO2 binds at the base of a hydrophobic channel at the interface of a hydrophobic pocket and the substrate binding site. Acetoacetate binds at an alternate anion binding site, as revealed in the bicarbonate and CoM disulfide bound structures. The enolate intermediate can be stabilized by an Ala430 carbonyl stabilized water molecule as revealed in the OHA bound structure, at a site different from that in KCoM bound structure. Together, the structures reveal a mechanism of concerted attack of a CO2 molecule on the enolate intermediate formed by the nucleophilic attack of Cys82 on the C-S bond of KCoM.en
dc.description.abstractAcetoacetate is stabilized at the alternate anion binding site, with the concomitant formation of a mixed disulfide. A nucleophilic attack by a water molecule on the mixed disulfide, accompanied by a weakening of interactions between CoM and Arg residues due to charge sharing with the acetoacetate carboxyl group, aids the release of CoM acetoacetate. Decarboxylation of acetoacetate occurs at the alternate anion binding site with the release of carboxyl group perhaps as bicarbonate. Structural refinement at 1.39Å in conjunction with density functional theory (DFT) optimization has been applied to address undefined aspects of the active site Hcluster that could impact the mechanism of reversible H2 oxidation by [FeFe] hydrogenase from Clostridium pasterianum. The model with highly accurate positioning of the H-cluster atoms was used for sequential structural optimization. The results of this optimization challenge the established paradigm that the dithiolate ligand bridging the Fe atoms of the 2Fe H-subcluster is a dithiomethylammonium and supports the assignment of a dimethyl ether.en
dc.language.isoengen
dc.publisherMontana State University - Bozeman, College of Letters & Scienceen
dc.subject.lcshHydrogenase.en
dc.subject.lcshDecarboxylases.en
dc.subject.lcshOxidoreductases.en
dc.titleStructure based mechanistic studies on 2-ketopropyl coenzyme M oxidoreductase / carboxylase from Xanthobacter autotrophicus and [FeFe] hydrogenase from Clostridium pasteurianum
dc.typeDissertation
dc.rights.holderCopyright Arti Sharma Pandey 2007en
thesis.catalog.ckey1290708en
thesis.degree.committeemembersMembers, Graduate Committee: Martin Lawrence (co-chair); Valerie Copie; Trevor Douglas; Brian Bothneren
thesis.degree.departmentChemistry & Biochemistry.en
thesis.degree.genreDissertationen
thesis.degree.namePhDen
thesis.format.extentfirstpage1en
thesis.format.extentlastpage187en
mus.identifier.categoryChemical & Material Sciences
mus.relation.departmentChemistry & Biochemistry.en_US
mus.relation.universityMontana State University - Bozemanen_US


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