Nitrogen Transfer in Biosynthetic Mechanisms 221 



stantiated in considerable detail, and the compound exhibits the chem- 

 ical properties to be expected of a substituted guanidine. Though 

 much more stable than the intermediate encountered in reaction lb, 

 when subjected to heat or dilute acid it rapidly undergoes non- 

 enzymatic conversion to a cyclic anhydride (amidine N to carboxyl C). 

 The latter can be converted back to argininosuccinic acid only by 

 exposure to dilute alkali. The two compounds behave like creatine 

 and creatinine in this respect and in solution tend to form equilibrium 

 mixtures governed by pH and temperature. The anhydride appears 

 to be metabolically inert. 18,19 



Reaction 4 merely serves to convert a disubstituted guanidine to a 

 monosubstituted one. The conversion is reversible and argininosuccinic 

 acid can be readily formed from arginine and fumaric acid by this 

 reaction. It involves but a small net change in free energy. The 

 formation and cleavage of this type of C-N bond cannot therefore 

 account for any significant part of the energy required for the synthesis 

 of urea. The energy utilization is confined to the C-N bond estab- 

 lished in reaction 3. The transformation of the ureide group to the 

 guanidine level represents the actual synthesis of the amidine group. 



Although phosphate-bond energy is utilized in the condensation and 

 orthophosphate is thereby liberated, it has not been possible to detect 

 a free, phosphorylated intermediate formed prior to condensation. It 

 may, of course, be formed transiently on the enzyme surface, or the 

 equilibrium of the reaction may be too unfavorable to allow detection. 

 For the present, the precise manner of phosphate-bond utilization is 

 a matter of conjecture, and the analogy to chemical guanidination, 

 mentioned above, is the basis for the hypothesis which now appears 

 most attractive. The isourea configuration, represented below in the 

 citrulline structure II, is the more reactive of two tautomeric forms. 



HoN H— N 



" \ \ 



C=0 ;=± C— OH 



/ / 



H— N H— N 



Hi R, 



i ii 



- P h 



H— N OH IT— N 



^. / Aspartic ^. 



C— O— P=0 — » C— NHR 9 



/ \ add / 



H— N OH H— N 



Ri Ri 



III IV 



