Inactivation of carbamoyl phosphate synthetase (ammonia) by elastase as a probe to investigate binding of the substrates

Abstract

Rat liver carbamoyl phosphate synthetase I is inactivated by elastase. Addition of ATP, Mg 2+, K + and N-acetyl-L-glutamate (the physiological allosteric activator) protects entirely, whereas acetylglutamate alone speeds inactivation. We have exploited these properties to investigate binding of these ligands. Acetylglutamate binds with low affinity (K D 0.25 mM) in the absence of other ligands, and with higher affinity (K D«0.1 mM) when ATP, Mg 2+ and K + are present. The apparent K D for ATP in the presence of acetylglutamate is intermediate between the K D values for the two ATP binding sites present in the enzyme; thus, binding of ATP to both sites is involved in protecting the synthetase. The data also indicate binding of MgATP and Mg 2+ in the absence of acetylglutamate. The results provide further evidence for conformational changes associated with allosteric activation of the enzyme.