The metabolism of propylene in Xanthobacter autotrophicus occurs via epoxypropane formation and subsequent metabolism by a three-step, four-enzyme pathway, utilizing the atypical cofactor Coenzyme M (CoM) to form acetoacetate. The last step in the epoxide carboxylase pathway is catalyzed by a distinctive member of the disulfide oxidoreductase (DSOR) family of enzymes, NADPH:2-ketopropyl CoM oxidoreductase/carboxylase (2-KPCC). 2-KPCC catalyzes the unorthodox cleavage of a thioether bond and successive carboxylation of the substrate. The focus of the research presented in this dissertation aims to elucidate the details of 2-KPCC that allow it to perform chemistry unconventional for typical DSOR members. Site-directed mutagenesis was used to mutate specific active site residues and to examine the catalytic properties of 2-KPCC upon these changes. Mutation of His137, the proximal histidine that directly coordinates the water molecule, eliminated essentially all redox-dependent activity of the enzyme while mutation of His84, the distal histidine that coordinates the water molecule through His137, diminished redox-dependent enzymatic activity to approximately 25% that of the wild type enzyme, confirming the respective roles of the histidine residues in stabilizing the enolate intermediate formed upon catalysis. Neither mutation of either histidine residue, nor mutation of either redox active cysteine residue had any negative effect on the rate of the redox-independent reaction catalyzed by 2-KPCC, the decarboxylation of acetoacetate. Mutation of Met140 resulted in an enzyme with drastically altered kinetic parameters and suggests Met140 plays a role in shielding the substrate from undesired electrostatic interactions with the surroundings.
The inhibitory properties of the structural CoM analogs, 2-bromoethanesulfonate (BES) and 3-bromopropanesulfonate (BPS), were examined and exploited to provide further detail on the active site microenvironment of 2-KPCC. Modification by BES results in a charge transfer complex between the thiolate of Cys87 and the oxidized flavin. The spectral features of this charge transfer complex have allowed the determination of the pK a of the Cys87 to be significantly higher than the flavin thiol in other DSOR enzymes. BPS has been shown to be a competitive inhibitor of 2-KPCC with an inhibition constant over two orders of magnitude lower than for that of BES.
|Advisor:||Ensign, Scott A.|
|Commitee:||Hevel, Joan, Hubbard, John, Seefeldt, Lance, Wolf, Paul|
|School:||Utah State University|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- Utah|
|Source:||DAI-B 72/12, Dissertation Abstracts International|
|Keywords:||Carboxylase, Coenzyme M, Ketopropyl, NADPH, Oxidoreductase|
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