Carbamoylphosphate synthase of rat liver has been purified to a homogeneous state. Its molecular weight is about 250,000.
When the catalytic activity is measured at 10°, using an optical method, an activation phase is detectable, which could be assigned to the N‐acetyl‐l‐glutamate induced transition of the enzyme from an inactive conformation to the active conformation. The half‐time of the process is about 1 min at 10 mM acetyl‐glutamate. The specific effect of the cofactor was found to be the labilization of the inactive conformation (as opposed to a stabilization of the active conformation).
At low temperatures the enzyme dissociates reversibly into probably two identical subunits. The dissociation seems to occur at the stage of the active conformation. ATP was found to hold back the dissociation by specifically stabilizing the active conformation.
The K,-values for ammonium and bicarbonate in carbamoylphosphate synthesis are 1.1 mM and 5.3 mM respectively a t saturation with ATP and acetylglutamate. The are independent of the concentration of the second substrate. The same K , for bicarbonate is found in the hydrolysis of ATP, which is catalyzed by the enzyme in the absence of ammonium. The [32P]orthophosphate-ATP-exchange, which is very slow, requires bicarbonate and ammonium.With these findings, the following mechanisms for the C-N-ligase step(s) of the enzyme are possible : (a) enzyme-bound carbamate is formed by a simultaneous reaction of ATP, bicarbonate and ammonium; (b) at first enzyme-bound "active CO," is formed in a rapid and reversible reaction, which releases both the ATP-products, ADP and orthophosphate only upon the reaction with ammonium. Possible structures for thia intermediate would be carboxyphosphate plus ADP and the addition compound of bicarbonate with ATP (with penta-coordinated phosphorus). The participation of biotin was excluded. As can be estimated from the kinetic findings, only a small fraction of the enzyme molecules would ever be charged with "active CO,"; the half-time of its decomposition, as calculated from the ATPase activity, would be I 2 sec.The following mechanism has been proposed by Jones [l] for the reaction of carbamoylphosphate synthetase of liver mitochondria, which requires N-acetyl-L-glutamate as an allosteric effector :The enzyme functions as a C-N-ligase and a phos- Biotin is not a constituent of the acetylglutamaterequiring enzyme from rat liver mitochondria, which is presently under investigation in our laboratory [7]. I n order to approach the problem of GO2-activation, we have made an analysis of the steady-state kinetics. The results, which have been summarized previously [8], do not fit reaction (a). They are consistent with an "active CO," that is formed from ATP and bicarbonate in a rapid reversible reaction and which contains both ATP-products, ADP and Pi. The kinetic situation is such, that the accumulation of the activation product and hence its chemical characterization appears infeasible. -The reversibility of the ligase-reaction could be demonstrated by the Pi-ATP-exchange reaction.
Laser induced fluorescence spectroscopy has been applied to measure the hyperfine structure and isotope shift of osmium I spectral lines in the visible range. Precise values for the hyperfine structure constants A and B of the most abundant odd mass isotope 189Os and the level isotope shift of altogether 43 excited energy levels were determined evaluating the measurements. Osmium in its natural abundance as well as an enriched sample of 189Os were used during the experiments. Additionally, a parametric analysis of the fine structure, the hyperfine structure and the isotope shift has been performed for the three even configurations 5d66s2, 5d76s and 5d8. Finally, the nuclear electric quadrupole moment Q for the isotope 189Os is determined to Q(5d66s2 = 0.98 (8) b for the configuration 5d66s2 and Q(5d76s = 0.97 (9) b for the configuration 5d76s.
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