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Analysis of the functional domains of the 1.3 S subunit of transcarboxylase

Magner, William John

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1994, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
Transcarboxylase is a complex, multi-subunit enzyme found in Propionibacterium shermanii which serves as a model for the family of biotin dependent carboxylases. The enzyme is composed of three subunits termed 12 S, 5 S and 1.3 S. The genes encoding these subunits are organized in an operon. They have been cloned and sequenced (Charles G. Thornton, Ph. D. dissertation August 1987). Computational analysis demonstrated a remarkable amount of sequence conservation among family members. This work describes E. coli expression systems for each of the genes found in this operon. The expressed recombinant proteins were studied to determine their multimeric structure and ability to catalyze the transcarboxylase partial and overall reactions. The recombinant forms of the 5 S and 12 S proteins were shown to be expressed and folded in apparently native dimeric and hexameric forms, respectively. Crude extracts of each expressed protein were shown to catalyze the appropriate transcarboxylase partial reaction.Additional studies focused on the 1.3 S biotinyl subunit and were directed toward understanding its structural and functional domains. The 1.3 S subunit was shown to be heat stable but sensitive to mutations which significantly shortened the subunit. Mutations were introduced into a pair of methionines at positions 88 and 90 which flank the biotinyl lysine in the 1.3 S sequence. The mutants were expressed, purified and assembled into enzyme complexes with the isolated 5 S and 12 S subunits for characterization. Mutation of the methionine residue at position 88 was shown to affect activity in all of the transcarboxylase reactions and the halflife of the carboxybiotinyl intermediate without altering the apparent activation energy of the carboxybiotin bond. The symmetric mutations of methionine 90 resulted in moderate changes in activity and carboxybiotin halflife but significantly altered the apparent activation energy of the carboxybiotin bond. We interpret these data to indicate that the methionine residue at position 90 interacts with the biotin or carboxybiotin and may have a catalytic function while the
David Samols (Advisor)
202 p.

Recommended Citations

Citations

  • Magner, W. J. (1994). Analysis of the functional domains of the 1.3 S subunit of transcarboxylase [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1061559282

    APA Style (7th edition)

  • Magner, William. Analysis of the functional domains of the 1.3 S subunit of transcarboxylase. 1994. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1061559282.

    MLA Style (8th edition)

  • Magner, William. "Analysis of the functional domains of the 1.3 S subunit of transcarboxylase." Doctoral dissertation, Case Western Reserve University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=case1061559282

    Chicago Manual of Style (17th edition)