Maintaining a proper balance of metabolites for cell growth requires regulation of biosynthetic pathways so that the formation of products is coupled to their consumption. Pyrimidine biosynthesis in Escherichia coli is controlled by changes in both the synthesis and catalytic activity of aspartate transcarbamoylase (ATCase). Understanding the molecular basis of the control of gene expression and the allosteric regulation of enzyme activity is a long-range objective of this research. Studies on the structure and interactions of wild-type ATCase and mutant forms can provide considerable insight regarding the mechanism of the allosteric transition of the enzyme from a low-affinity, constrained conformation to a relaxed form having a high affinity for substrates. How rapidly this conformational change occurs will be measured with hybrid molecules containing spectral probes located on regulatory chains which sense the propagation of perturbations resulting from ligand binding to catalytic chains. Also models for the allosteric transition can be tested by studies of the reverse reaction catalyzed by ATCase. Because this rate is enhanced 40-fold by limited inhibitor binding, the number of active sites generated by the binding of only one molecule of inhibitor to the enzyme can be determined directly by kinetic titration. Manipulation of a plasmid containing the two linked structural genes, pyrB and pyrI, encoding the catalytic and regulatory chains yields a DNA fragment which causes the synthesis of active catalytic subunits devoid of regulatory properties. Physiological studies should provide valuable information regarding the in vivo role of allostery. Uncoupling the genes in the pyrBpyrI operon and altering the amounts of the separate RNA transcripts will facilitate analysis of ATCase assembly in vivo. Application of a technique for the positive selection of mutant strains defective in enzyme activity yielded 15 inactive missense mutants whose alterations will be determined from nucleotide sequences. X-ray diffraction and NMR studies should reveal the effects of amino acid substitutions of folding of the polypeptide chains. Knowledge of the tertiary and quaternary structures of inactive mutants, coupled with studies of ligand-promoted conformational changes in wild-type catalytic subunits, will provide information about the mechanism of catalysis. Hybridization experiments with defective mutants can lead to structural models which account for the in vivo restoration of biological activity in complementation studies. These studies can provide insight into malfunctions in metabolic control.
| 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 |
