ASSOCIATION OF THE CATALYTIC SUBUNIT OF ASPARTATE TRANSCARBAMOYLASE WITH A ZINC-CONTAINING POLYPEPTIDE FRAGMENT OF THE REGULATORY CHAIN LEADS TO INCREASES IN THERMAL-STABILITY

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stabili ty to the manifold interchain interactions among the 12 polypeptide ch ains. With the availability of a recombinant 70-amino acid zinc-contai ning polypeptide fragment of the regulatory chain of ATCase, it has be come possible to analyze directly the interaction between catalytic an d regulatory chains in a complex of simpler structure independent of o ther interactions such as those between the 2 C trimers, which also co ntribute to the stability of the holoenzyme. Also, the effect of the i nteraction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured d irectly. Differential scanning microcalorimetry experiments demonstrat ed that the binding of the zinc domain to the C trimer leads to a comp lex of markedly increased thermal stability. This was shown with a ser ies of mutant forms of the C trimer, which themselves varied greatly i n their temperature of denaturation due to single amino acid replaceme nts. With some C trimers, for which t(m) varied over a range of 30 deg rees C due to diverse amino acid substitutions, the elevation of t(m) resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of t(m) for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed w hen the holoenzymes containing the mutant C trimers were subjected to heat denaturation. In an extreme case with a mutant form involving rep lacement of Glu 86 by Ala in the catalytic chains, this was manifested by a change in t(m) for the trimer of 44.6 degrees C to 64.6 degrees 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 t(m) than isolated C trim er. The effect of the zinc domain on the t(m) of the complex with C tr imer 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 .