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 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)
Peterson
C B
CB
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Zhou
B B
BB
Hsieh
D
D
Creager
A N
AN
Schachman
H K
HK
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*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D001221:Aspartate Carbamoyltransferase; D015032:Zinc