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  • Title: Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis.
    Author: Stevens RC, Chook YM, Cho CY, Lipscomb WN, Kantrowitz ER.
    Journal: Protein Eng; 1991 Apr; 4(4):391-408. PubMed ID: 1881865.
    Abstract:
    Crystal structures are known for aspartate carbamoyltransferase (ATCase) in the T and R states, with and without the allosteric activator adenosine triphosphate (ATP) or inhibitor cytidine triphosphate (CTP). Visual inspection of X-ray crystal structures does not provide all of the information necessary for the determination of structure--function relationships in protein molecules. This problem is compounded because the crystalline states of the molecule may introduce effects due to crystal packing, restricted flexibility and less than optimum enzymatic conditions. Therefore, alternative techniques are required to test mechanisms conjectured from three-dimensional crystal structures of proteins. The technique of site-specific mutagenesis allows the researcher to test structure--function models based on three-dimensional structures and to obtain further insight into characteristics of the enzyme. Site-specific mutagenesis has been used to probe residues believed to be critical in the structure and function of ATCase. Selection of residues to be mutated has depended extensively on three-dimensional crystal structures of the enzyme. To date, 48 site-specific mutations at 37 different amino acid sites have been published. Although a total of 118 mutants at 58 different sites has been communicated to our laboratory, only published mutants will be considered in this review. In this paper, we compile for the first time, review, and analyze the site-specific mutants of ATCase. Site-specific mutagenesis of proteins has become a powerful technique in modern-day molecular biology, especially in studying a molecule as large as aspartate carbamoyltransferase. In this review, the role of site-specific mutagenesis of ATCase is discussed and improvements in the analysis are suggested.
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