Half-of-the-Sites Reactivity and the Conformational States of Cytidine Triphosphate Synthetase

The affinity label 6-diazo-5-oxonorleucine (DON) reacts with only half of the total glutamine sites of CTP-synthetase, even though the subunits are identical. The binding of DON to half of the subunits abolishes the glutamine activity of the enzyme; thus, vacant active sites as well as occupied sites are turned off. However, the DON-labeled enzyme retains its overall activity with ammonia and its characteristic binding of other ligands such as ATP, UTP, and GTP. It is postulated that DON labeling is an example of extreme or absolute negative cooperativity in which the conformational change induced by DON in the covalently labeled subunit is transmitted to the neighboring subunit so that no DON reaction can occur there. This model provides a general explanation for other enzymes in which half-of-the-sites reactivity has been observed. The fact that the DON-induced change can abolish glutamine activity while leaving ammonia activity and nucleotide binding unaffected indicates that the protein is designed to allow specific transmission of conformational changes.