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Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Summary
Circularly permuted aspartate transcarbamoylase (ATCase) catalytic chains fold and assemble into active trimers. Enzyme activity and stability are independent of N- and C-terminal positions, highlighting sequence-driven protein folding.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Active enzyme formation from polypeptide fragments of aspartate transcarbamoylase (ATCase) has been demonstrated.
- Wild-type ATCase catalytic chains exhibit proximity of their NH2 and COOH termini.
Purpose of the Study:
- To investigate if circularly permuted catalytic chains of ATCase can fold and assemble into active trimers.
- To explore the role of N- and C-terminal positions in protein folding and oligomer assembly.
Main Methods:
- Construction of altered genes encoding circularly permuted ATCase catalytic chains.
- Expression and in vivo association of permuted chains into active trimers.
- In vitro assembly of permuted trimers with wild-type regulatory dimers.
Main Results:
- Circularly permuted ATCase chains expressed well and formed active trimers in vivo.
- These trimers combined with regulatory dimers to form ATCase-like molecules.
- Enzymes with permuted chains showed similar Vmax and stability but higher Km and lacked allosteric properties compared to wild-type.
Conclusions:
- Protein folding into defined domains and assembly of stable oligomers are not dependent on the location of N- and C-termini.
- The final amino acid sequence dictates folded structures, not the order of amino acid addition.