Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase)

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX₁X₂RKX₃GQ- ethylenediamine-2,4-dinitro-phenyl, where P₂, P₂', and P₃ residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P₁ and P₁' residues, and adjacent residues are designated P₂, P₃, P₂', P₃', etc.) there was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P₂ residues having the greatest effect. The S₃, S₂, and S₂' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P₂ position, were among the best P₂' residues. The specificity at P₃ was generally opposite that of P₂. Residues at P₂, and to a lesser extent at P₃, influenced the cleavage site. At the P₂ position, his, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P₂ Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P₁' or P₁ position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.