Aspartate transcarbamoylase (ATCase) of E. coli has become the paradigm for scientists attempting to understand the control of metabolic processes through the action of regulatory enzymes. As in the case of hemoglobin, much has been learned through the application of a proposed model for the allosteric transition whereby the protein is converted from a low-affinity (and low- activity) compact (taut) T state to a high activity, relaxed R conformation. Whereas the structure of the T state is readily available through crystallographic studies, the R conformation is not accessible since the equilibrium between these two states is predominantly in favor of the T state. Only with mutant forms of the enzyme is it possible to shift the population of molecules in the two states toward the R conformation. Recent structural work on the catalytic subunit of ATCase in the absence and presence of an active site ligand indicates that the widely proposed, and tacitly accepted, structure of the ATCase-ligand complex as representing the R conformation should be questioned. Hence extensive crystallographic studies will be conducted on three mutant enzymes that appear to be excellent candidates for determining the R state structure. Similar x-ray diffraction experiments will be performed on a simple model of ATCase composed of one catalytic trimer and three fragments of the regulatory polypeptide chain known as the zinc domain. This complex, which is much smaller than ATCase, has the characteristics of the intact enzyme in the R state. Thus its structure would provide invaluable information of the detailed conformation of the active sites in the R state. One of the mutants used for these studies shows that relatively small changes in pH lead to a conversion of the T to the R state. The rate of this interconversion will be measured using the synchrotron so that kinetic data for the T yields R and the R yields T conversions can supplement equilibrium studies. The development in this laboratory of a technique for random circular permutation of genes and the expression of polypeptide chains has provided new opportunities to relate in vivo folding of proteins to the in vitro experiments being conducted so widely. Through detailed understanding of the allosteric transition and the folding of polypeptide chains to give functional proteins, our knowledge of regulatory enzymes, and pyrimidine biosynthesis in particular, will be greatly enhanced. Such studies will contribute toward circumventing aberrations in metabolic control processes.
| Endrizzi, James A; Beernink, Peter T (2017) Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes. Protein Sci 26:2221-2228 |
