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In vivo assembly of aspartate transcarbamoylase from fragmented and circularly permuted catalytic polypeptide chains

Department of Molecular and Cell Biology and Virus Laboratory, University of California at Berkeley, Berkeley, California 94720, USA

Reprint requests to: Howard K. Schachman, University of California at Berkeley, Department of Molecular and Cell Biology, 229 Stanley Hall #3206, Berkeley, California 94720-3206, USA; e-mail: schach{at}socrates.berkeley.edu; fax: (510) 642-8699.

Previous studies on Escherichia coli aspartate transcarbamoylase (ATCase) demonstrated that active, stable enzyme was formed in vivo from complementing polypeptides of the catalytic (c) chain encoded by gene fragments derived from the pyrBI operon. However, the enzyme lacked the allosteric properties characteristic of wild-type ATCase. In order to determine whether the loss of homotropic and heterotropic properties was attributable to the location of the interruption in the polypeptide chain rather than to the lack of continuity, we constructed a series of fragmented genes so that the breaks in the polypeptide chains would be dispersed in different domains and diverse regions of the structure. Also, analogous molecules containing circularly permuted c chains with altered termini were constructed for comparison with the ATCase molecules containing fragmented c chains. Studies were performed on four sets of ATCase molecules containing cleaved c chains at positions between residues 98 and 99, 121 and 122, 180 and 181, and 221 and 222; the corresponding circularly permuted chains had N termini at positions 99, 122, 181, and 222. All of the ATCase molecules containing fragmented or circularly permuted c chains exhibited the homotropic and heterotropic properties characteristic of the wild-type enzyme. Hill coefficients (n_H) and changes in them upon the addition of ATP and CTP were similar to those observed with wild-type ATCase. In addition, the conformational changes revealed by the decrease in sedimentation coefficient upon the addition of a bisubstrate analog were virtually identical to that for the wild-type enzyme. Differential scanning calorimetry showed that neither the breakage of the polypeptide chains nor the newly formed covalent bond between the termini in the wild-type enzyme had a significant impact on the thermal stability of the assembled dodecamers. The studies demonstrate that continuity of the polypeptide chain within structural domains is not essential for the assembly, activity, and allosteric properties of ATCase.

Keywords: Circular permutation; cooperativity; folding; fragment complementation; protein engineering; stability

Abbreviations: ATCase, aspartate transcarbamoylase • C, catalytic trimer or subunit • c, catalytic polypeptide chain • R, regulatory dimer or subunit • r, regulatory polypeptide chain • wt as subscript, wild type • fr as subscript, fragmented polypeptide chain • cp as subscript, circularly permuted • c and number following it in subscript designate the position of the amino acid residue in the wild-type catalytic chain at which the new N terminus in the fragmented or circularly permuted chain is located • H6 as subscript, hexa-His sequence at the N terminus of the regulatory chain • MOPS, 3-(N-morpholino)propanesulfonic acid • PAGE, polyacrylamide gel electrophoresis • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis • PCR, polymerase chain reaction • PALA, N-(phosphonacetyl)-L-aspartate • T_m, melting temperature corresponding to the maximum temperature in the endotherm obtained by differential scanning microcalorimetry

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