Mitochondrial Carbamoyl Phosphate Synthetase Activity in the Absence of N‐Acetyl‐l‐glutamate

Abstract

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. K , values for ATP, Mg" and K + are greatly increased, the K , for HCO; is not changed much, and the K , for NH: is markedly reduced. V,,, for the synthetase reaction is < 20 % of that of the acetylglutamateactivated enzyme whereas VmaX for the ATPase activity is >40% of that with acetylglutamate. Pulse-chase experiments with H14CO; show formation of less "active C02" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20 -30 pM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATP, (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 pM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time.From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATP, is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the y-phosphoryl group of ATP, appears to be facilitated.It has been known for many years that carbamoylphosphate synthetase of liver mitochondria requires acetylglutamate or an analogue for activity, but the role of this cofactor in the reaction has remained unclear [l]. Recently we have shown that a variety of cryoprotectants' (e.g. dimethylsulphoxide, ethyleneglycol, glycerol and sucrose) partly replace acetylglutamate in activating theenzyme [ 2 ] . The activation by such a range of chemical agents and the lack of evidence for direct participation of acetylglutamate in the enzyme reaction are strong indications that the cofactor and the cryoprotectants activate by an allosteric mechanism. If the mechanism of activation is allosteric the enzyme should possess some activity in the absence of the activator and the kinetics of the nonactivated enzyme should provide information about the activating process. Indeed, although no activity was detected with the frog enzyme [l], about 2 % of the activity has been reported with the rat enzyme in the absence of acetylglutamate' [3]. However, the kinetics of the enzyme in the absence I The term 'cryoprotectants' is applied to various agents that protect cells from the effects of freezing and to those agents that protect cold-labile proteins, such as carbdmoyl-phosphate syntethase 1161. Such compounds are known to stabilize proteins and are frequently added for this purpose rather than to protect specifically against ...

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

“…We have calculated from these results a Kd 2 2.5 mM for acetylglutamate. We also found [5] that incubation of the rat enzyme in the absence of ATP with 10 mM, but not with 50 pM, acetylglutamate, decreased the time-lag in subsequent assays ; Squares and circles represents inactive (T) and active (R) forms of the enzyme. tt represents steps at equilibrium when there is no NH3.…”

“…At the concentrations of ATP (0.55-5.6 mM) shown here to have an effect on the Kd for acetylglutamate, the binding site for ATP, is saturated (Kd for ATPB % 20 pM, with or without acetylglutamate [5]). Thus, binding of ATPA, which occurs with lower affinity (& for ATPA > 0.2 mM [5,15]) is required for acetylglutamate binding.…”

“…It is not known whether K t and Mgz+ are released with each catalytic cycle; however, for simplicity, the free enzyme i s shown to be regenerated. For further details, see [5] thus, Kd for acetylglutamate in the absence of ATP is very large. Binding with such low affinity exceeds the detection limit in our assays.…”

“…Thus, binding of ATPA, which occurs with lower affinity (& for ATPA > 0.2 mM [5,15]) is required for acetylglutamate binding. In agreement with this conclusion, omission of acetylglutamate greatly decreases the affinity of the enzyme for ATPA [S].…”

“…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 …”