title: Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.
abstract: The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720.
authors.forename: C B
authors.initials: CB
authors.lastname: Peterson
authors.forename: B B
authors.initials: BB
authors.lastname: Zhou
authors.forename: D
authors.initials: D
authors.lastname: Hsieh
authors.forename: A N
authors.initials: AN
authors.lastname: Creager
authors.forename: H K
authors.initials: HK
authors.lastname: Schachman
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D001221:Aspartate Carbamoyltransferase; D015032:Zinc