Role of the 200's loop in homotropic and heterotropic responses of Escherichia coli aspartate transcarbamylase /
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1989.
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| Subjects: | |
| Online Access: | ProQuest, Abstract Link to OAKTrust copy |
| Abstract: | In this study, amino acids within the 200's region of the equatorial domain of Escherichia coli aspartate transcarbamylase (ATCase) have been shown to be involved in various kineti phenomena. The structure/function assignments of specify residues were based on comparisons of amino acid divergence from various enteric bacteria. The technique of site-directed mutagenesis was applied to selected amino acids which differ from one species to another. The results from the analysis of the substitution of aspartate 203c (catalytic chain) and tryptophan 209c with glutamate and tyrosine, respectively, demonstrated that the mutant holoenzyme possessed an improved catalytic efficiency (the V[max] increased from 14.4 μmoles/hr/μg). This enhanced activity was also observed for the catalytic subunits, with values increasing from 26.9 μmoles/hr/μg for wild-type ATCase to 48.1 μmoles/hr/μg for the mutant enzyme. A mutational substitution within the regulatory polypeptide, arginine 130 (Arg 130r) to alanine, resulted in an enzyme lacking CTP + UTP synergistic effects with only slight (5%) inhibition by CTP. The ATP effect was similar to that observed in the wild-type enzyme. This substantiates the hypothesis that separate structural channels are involved in the transmission of the ATP and CTP effects. An analysis of a substitution at position 204c in which glutamate was replaced by alanine revealed a possible loss of stability in the Instate of the enzyme. Both the mutant holoenzyme and the separated catalytic subunits possessed a 1000-fold decrease in maximal activity. A mutation at position 200c in which aspartate is replaced by lysine produced an enzyme with an [S][0.5] for asparate of 70mM, compared to the wild-type value of 6.2mM. In spite of this reduced affinity, normal effector responses were observed in this mutation. This study demonstrated that several amino acids localized within a small region (residues 192-212) of the equatorial domain of E. coli ATCase influence both homotropic and heterotropic responses of the enzyme. The localization of small, discrete regions with specific functions is an important step in the elucidation of regulatory and catalytic specificities in this enzyme. |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Biochemistry." |
| Physical Description: | x, 99 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |