Recognition of alpha-amino acids bearing various C=NOH functions by nitricoxide synthase and arginase involves very different structural determinants

Several alpha-amino acids bearing a C=NOH function separated from the C alp ha carbon by two to five atoms have been synthesized and tested as substrat es or inhibitors of recombinant nitric oxide synthases (NOS) I and II and a s inhibitors of rat liver arginase (RLA). These include four N-hydroxyguani dines, N-omega-hydroxy-L-arginine (NOHA) and its analogues homo-NOHA, nor-N OHA, and dinor-NOHA, two amidoximes bearing the -NH-C(CH3)=NOH group, and t wo amidoximes bearing the -CH2-C(NH2)= NOH group. Their behavior toward NOS and RLA was compared to that of the corresponding compounds bearing a C=NH function instead of the C=NOH function. The results obtained clearly show that efficient recognition of these cr-amino acids by NOS and RLA involves very different structural determinants. NOS favors molecules bearing a -NH- C(R)=NH motif separated from C alpha by three or four CH2 groups, such as a rginine itself, with the necessary presence of delta-NH and omega-NH groups and a more variable R substituent. The corresponding molecules with a C=NO H function exhibit a much lower affinity for NOS. On the contrary, RLA best recognizes molecules bearing a C=NOH function separated from C alpha by th ree or four atoms, the highest affinity being observed in the case of three atoms. The presence of two omega-nitrogen atoms is important for efficient recognition, as in the two best RLA inhibitors, N-omega-hydroxynorarginine and N-omega-hydroxynorindospicine, which exhibit IC50 values at the microm olar level. However, contrary to what was observed in the case of NOS, the presence of a delta-NH group is not important. These different structural r equirements of NOS and RLA may be directly linked to the position of crucia l residues that have been identified from crystallographic data in the acti ve sites of both enzymes, Thus, binding of arginine analogues to NOS partic ularly relies on strong interactions of their delta-NH and omega-NH2 groups with glutamate 371 (of NOS LI), whereas binding of C=NOH molecules to RLA is mainly based on interactions of their terminal OH group with the binucle ar Mn(II)... Mn(II) cluster of the enzyme and on possible additional bonds between their omega-NH2 group with histidine 141, glutamate 277, and one Mn (II) ion. The different modes of interaction displayed by both enzymes depe nd on their different catalytic functions and give interesting opportunitie s to design useful molecules to selectively regulate NOS and arginase.