Association of the catalytic subunit of aspartate transcarbamoylase with a zinc‐containing polypeptide fragment of the regulatory chain leads to increases in thermal stability

Cynthia B. Peterson, Bin‐Bing ‐B Zhou, Durwynne Hsieh, Angela N.H. Creager, H. K. Schachman

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8 Scopus citations


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 °C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 °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. In an extreme case with a mutant form involving replacement of Glu 86 by Ala in the catalytic chains, this was manifested by a change in tm for the trimer of 44.6 °C to 64.6 °C for the holoenzyme. These results contribute to our understanding of an earlier observation that scanning calorimetry on wild‐type ATCase gave 2 transitions, with the high temperature peak, which is assigned to melting of C trimers, exhibiting a higher tm than isolated C trimer. The effect of the zinc domain on the tm of the complex with C trimer provides an explanation for this increase in thermal stability, i.e., during heat denaturation of the holoenzyme, the C trimer is still associated with the folded zinc domain fragments of regulatory chains.

Original languageEnglish (US)
Pages (from-to)960-966
Number of pages7
JournalProtein Science
Issue number6
StatePublished - Jun 1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry


  • conformational change
  • differential scanning microcalorimetry
  • enhanced thermal stability
  • interchain interaction
  • zinc domain


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