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