From Biological Diversity to Structure-Function Analysis: Protein Engineering in Aspartate Transcarbamoylase
The largest of these units are discrete polypeptides or superdomains within multifunctional proteins which have been shown to be uniquely involved in specific catalytic steps within the CAD or CA complexes of eukaryotic pyrimidine biosynthesis.
The catalytic polypeptides of various ATCases are organized into two discrete and separable binding domains for its substrates, carbamoyl phosphate and aspartate.
The regulatory polypeptides of the enteric bacterial enzymes also contain two discrete tertiary domains, the Allosteric Binding Domain and Cys 4 coordinated Zinc Domain involved in the protein:protein interface between the regulatory and catalytic polypeptides of the holoenzyme.
There are sub-domain structural units within the various polypeptides which have a coordinated impact on specific catalytic and regulatory functions in the enzyme.
Finally, it has been possible to ascribe some individual function to specific amino acids relative to ligand binding, zinc coordination, protein:protein interactions, and the structural reorganizations in the T-R transition of the enteric holoenzymes.
Comparisons of the catalytic sequences of various ATCases have revealed substantial conservation of primary and predicted secondary structures. Based upon the sequence alignment and the E. coli ATCase crystal structure, the hamster ATCase superdomain tertiary structure has been modeled with interactive computer graphics. The predicted conservation of structure/function relationships were verified experimentally through the construction of active catalytic bacterial/hamster chimeric enzymes. It has also been possible to verify the apparent structural homologies of ATCase and ornithine transcarbamoylase (OTCase, 188.8.131.52), the parallel enzyme from arginine biosynthesis, by exchange of the amino acid binding domains of the two enzymes. Extending these observations and protein engineering philosophies into the formation of hybrid and chimeric enzymes has resulted in the production of ATCases with altered catalytic and regulatory characteristics.
KeywordsRegulatory Subunit Pyrimidine Biosynthesis Chimeric Enzyme Zinc Binding Domain Hybrid Enzyme
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