Acetyl glutamate

The rate of urea formation is mainly controlled by reaction [1]. N-acetyl glutamate, as an allosteric effector, activates carbamoyl-phosphate synthase. In turn, the concentration of acetyl glutamate depends on arginine and ATP levels, as well as other factors. 

Regulators Activator N-acetyl- glutamate Inhibitor UTP Activator ATPiWfi ... 

Acetyl Glutamate-Induced Activation and Inactivation of Carbamyl-P Synthetase... 

The investigation of the mechanism of action of carbamyl-P synthetase has been always associated with the study of the role of acetyl glutamate or related cofactors in the reaction ... 

In extension of previous observations from this laboratory (20) Metzen-berg, Marshall, and Cohen (32) demonstrated that the frog liver car-bamyl-P synthetase can be activated by preincubation with acetyl glutamate. The activation phenomenon has been extensively studied in our laboratory. 

Table III. Effect of Length of Preincubation on Activation of Frog Liver Carbamyl-P Synthetase by Acetyl Glutamate. (37)...

Acetyl Glutamate Present during Preincubation Preincubation ... 

This relatively rapid type of activation explains the autocatalytic curves obtained when initial rates of citrulline synthesis are compared with those obtained after several minutes incubation. Table IV demonstrates that by increasing the acetyl glutamate concentration and the length of incubation for the over-all synthesis, the activation was obscured. This is why the activation effect remained undetected until extremely short incubation times and low temperatures were used. Bringing a complicated reaction mixture to incubation temperature, waiting for equilibration, and completing with a last component, as is often done, may obscure effects such as the ones described here. 

In the experiments of Table IV 3.2 mg. of protein from frog liver preparations (35), 100 /mmoles of Tris-Cl" at pH 7.4, and the indicated amount of acetyl glutamate, in a volume of 1 ml., were preincubated at 38° for 5 minutes. The tubes were cooled to 25° and completed, at definite intervals, with the components and concentration of reagents indicated in Table HI, and then incubated at 25° for the indicated length of time. 

Figure 2. Dependence of activation phenomenon on concentration of acetyl glutamate...

Figure 3. Influence of pH on activation of frog liver carbamyl-P synthetase by acetyl glutamate...

Cold Lability of Carbamyl-P Synthetase. The acetyl glutamate-activated enzyme is less stable at low temperatures than the nonacti-vated enzyme preparations (35). The frog enzyme is stable over a wide temperature range. In the presence of acetyl glutamate, it retains its stability if stored above 10° and as high as 45°, but it is inactivated atO to 5°. This is one of four cases known to us, of enzymatically active... 

Acetyl Glutamate Present during Storage None 1 x 10 M... 

The prompt removal of acetyl glutamate from the preincubation mixtures, by dialysis at room temperature or by precipitation of the protein with ammonium sulfate, prevented the cold inactivation. The presence of ammonium sulfate, in concentrations as high as 0.17M, did not protect the enzyme against cold inactivation (at higher molarities of the salt, precipitation began to occur). Once the cold inactivation had taken place, the enzymatic activity could not be restored either by precipitation with ammonium sulfate, or by reincubation with acetyl glutamate or with ATP and Mg+2. 

Top. Sedimentation pattern (left to right) of frog liver car bamyl-P synthetase (35) containing 5.5 mg. of protein per ml. after incubation at 38°, for 5 minutes Bottom. Same preparation made 0.01M in acetyl glutamate, incubated at 38°for 5 minutes, and stored at 0 °for 48 hours... 

Other Physical Measurements. Viscosity measurements did not show differences between samples treated with acetyl glutamate and controls, before and after storage in the cold (35). Extensive series of ultracentrifuge measurements did not clarify the phenomenon the latter studies were conducted at several protein and acetyl glutamate concentrations, and at temperatures varying from 5° to 20° only after cold inactivation for 40 hours were marked changes noticeable. 

A Enzyme in 0.005M acetyl glutamate O Enzyme in 0.005M acetyl glutamate after cold in-. activation... 

The addition of 1 x 10 3 M acetyl glutamate to frog liver enzyme preparation causes a 10% and immediate increase in fluorescence. This effect has been confirmed by Edelhoch (8), who concurs in our interpretation that this finding signifies acetyl glutamate-induced conformational changes of carbamyl-P synthetase. We believe that the decrease in stability induced by substrates and cofactors is, whenever it is found to occur, the simplest and most sensitive method available for the detection and study of conformational changes in enzymic proteins. 

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