Studies on the Action of Hydrazine, Hydroxylamine, and Other Amines in the Carbamyl Phosphate Synthetase Reaction

The carbamyl phosphate synthetase of frog liver particles catalyzes the synthesis of 1 mole of carbamyl phosphate, 1 mole of orthophosphate, and 2 moles of adenosine diphosphate from ammonia, bicarbonate, and 2 moles of ATP. It can produce orthophosphate from ATP if ammonia is replaced by either hydroxylamine or hydrazine. Both hydroxylamine and hydrazine are competitive inhibitors of carbamyl phosphate synthesis and, therefore, presumably replace ammonia or decompose the activated carbonate which is a precursor of carbamyl phosphate. Hydrazine replaces ammonia because it is incorporated into a labile phosphate which appears to be N-aminocarbamyl phosphate. It can be shown that the new derivative contains hydrazine, 14C from 14C-bicarbonate, and phosphate, the latter two in equal amounts. N-Aminocarbamyl phosphate migrates more slowly toward the anode at pH 6 in an electrical field than carbamyl phosphate. It is a markedly more acid-labile and base-stable phosphate than carbamyl phosphate. N-Aminocarbamyl phosphate cannot serve as a substrate for ornithine transcarbamylase. Hydroxylamine, however, does not appear to yield a carbamyl phosphate or hydroxylamine phosphate. It can be shown that hydroxylamine can act catalytically, and it is therefore suggested that it decomposes the activated carbonate which is the product of the first mole of ATP utilized by the enzyme. If 1 or 2 of the hydrogen atoms of hydrazine or hydroxylamine are replaced with methyl groups, the derivatives are not substrates. Glutamine will not serve as a substrate.