A combined kinetic and modeling study of the catalytic center subsites of human angiogenin

Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B₁ pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B₁. Although the B₂ site that preferentially binds purines on the 3' side of B₁ is also weak, its associated phosphate subsites make substantial contributions: both 3',5'-ADP and 5'-ADP have K(i) values 6-fold lower than for 5'-AMP, and adding a 3'- phosphate to the substrate CpA increases k(cat)/K(m) by 9-fold. Thus Ang has a functional P₂ site on the 3' side of B₂ and a site for a second phosphate on the 5' side of B₂. Modeling of an Ang-d(ApTpApA) complex suggested that Arg-5 forms part of the P₂ site and that a 2'-phosphate might bind more tightly than a 3'-phosphate. Both predictions were confirmed kinetically. The subsite map obtained by this combined approach indicated that 5'- diphosphoadenosine 2'-phosphate might be a more potent inhibitor than any of the nucleotides tested thus far. Indeed, its K(i) value of 150 μM is 50- fold lower than that for the best nucleotide previously reported and 400- fold lower than the K(m) for the best dinucleotide substrate. This compound may serve as a suitable starting point for the eventual design of tight- binding inhibitors of Ang as antiangiogenic agents for human therapy.