Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia)

Abstract

The binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer ( M , 165 000). K ', Mg2 + (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO; was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K f and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCOY, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO; ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher K , for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. A D P or carbamoyl phosphate alone did not promote substantial binding.Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast.A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. N-Acetyl-L-glutamate activates the mitochondria1 carbarnoyl-phosphate synthetase from ureotelic animals. This activator appears to play a key role in regulating the rate of carbamoyl phosphate production and thus of urea synthesis [I 1. Although the need for acetylglutamate was recognized early [2], its exact role has remained unclear for many years. Tests to demonstrate a direct participation of acetylglutamate in the reaction have always been negative. On the basis of this and of other indirect evidence it was suggested that acetylglutamate plays an allosteric role [3]. We provided strong support for this view by demonstrating that several cryoprotectants, unrelated to acetylglutamate, partly replace this agent [4]. As expected from an allosteric mechanism of activation, we also found enzyme activity in the absence of acetylglutamate and of cryoprotectants, and we compared the kinetics of the reaction in the absence and in the presence of acetylglutamate. From these and other results we have proposed a model for allosteric activation of the enzyme [5].To understand better the mode of action of acetylglutamate, information on binding of this cofactor would be

Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene

“…They also observed that both Mg2+ and ATP are required for binding of N-acetylglutamate to the enzyme. Their studies are described in the accompanying paper [35].…”

“…Rubio and coworkers [35] also recently established the conditions for binding of N-['4C]acetylglutamate to carbamoyl-phosphate synthetase, using the purified enzyme. They also observed that both Mg2+ and ATP are required for binding of N-acetylglutamate to the enzyme.…”

Limited proteolysis reveals low‐affinity binding of N‐acetyl‐L‐glutamate to rat‐liver carbamoyl‐phosphate synthetase (ammonia)

“…We have previously demonstrated, using rate of dialysis and ultracentrifugation [7], that the enzyme binds one molecule of [14C]acetylglutamate with high affinity (Kd % 0.6 -1 pM) when K + and Mgz+ (which are essential ionic activators of the enzyme) and a mixture of either ADP and carbamoyl phosphate or ATP and HCO; are present. HCO; appears not to be essential, although it increases the affinity substantially.…”

“…We have previously demonstrated, using rate of dialysis and ultracentrifugation [7], that the enzyme binds one molecule of [14C]acetylglutamate with high affinity (Kd % 0.6 -1 pM) when K + and Mgz+ (which are essential ionic activators of…”

Isomerization of the free enzyme versus induced fit: effects of steps involving induced fit that bypass enzyme isomerization on flux ratios and countertransport

“…However, the activator for rat carbamoylphosphate synthetase, acetylglutamate, has been shown to remain in rapid exchange with the solution despite a slow activation process taking many minutes that is associated with a very marked increase in affinity of the enzyme for acetylglutamate [31,32].…”