Biosynthesis of urea; enzymatic mechanism of arginine synthesis from citrulline.

In urea synthesis, according to the Krebs-Henseleit ornithine cycle (l), the transfer of nitrogen to citrulline to form arginine (Step II) was thought to occur from NH$. It was not until Cohen and Hayano (2) succeeded in demonstrating rapid arginine synthesis in liver homogenates from glutamic acid and citrulline that the transfer from an amino acid rather than from NH, was recognized as a main pathway in urea formation. Under their conditions, arginine was formed in the presence of oxygen, Mg++, and catalytic amounts of adenosine triphosphate (ATP). This was the same reaction observed earlier by Borsook and Dubnoff (3) in kidney slices and considered at the time to be a transamination with simultaneous oxidative removal of 2 H atoms (transimination). An ar-keto acid, supposedly the second product of the reaction, would of course escape detection in respiring preparations. Whereas in the kidney slice experiments of Borsook and Dubnoff, both aspartic and glutamic acid were equally effective as amino group donors, Cohen and Hayano found in liver homogenates that glutamic acid was about 4 times as effective as aspartic acid. They concluded therefore that glutamic acid was the specific donor in the Borsook-Dubnoff reaction, aspartic acid being utilized only to the extent that it could be converted to glutamic acid. This investigation was undertaken to elucidate further some fundamental aspects of the enzymatic mechanism involved. As briefly reported earlier, it has been possible to obtain arginine synthesis with a partially purified enzyme system prepared by alcohol fractionation of acetone powder extracts of mammalian liver (4). In the isolated system, aspartic acid and citrulline are converted to arginine and malic acid in the presence of Mg++. ATP participates directly as a reactant and the reaction proceeds anaerobically without involving the transport of H atoms. In view of the evidence presented below, the transfer