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

The subsite specificity of rat nardilysin was investigated using fluorogeni c substrates of the type 2-amino-benzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine -2,4-dinitrophenyl, where P-2, P-2', and P-3 residues were varied. (The nom enclature of Schechter and Berger (Schechter, L, and Berger, A. (1967) Bioc hem, Biophys, Res. Commun, 27, 157-162) is used where cleavage of a peptide occurs between the P-1 and P-1' residues, and adjacent residues are design ated P-2, P-3, P-2', P-3', etc.) There was little effect on II, among diffe rent residues at any of these positions, In contrast, residues at each posi tion affected k(cat), with P-2 residues having the greatest effect, The S-3 , S-2, and S-2' subsites differed in their amino acid preference, Tryptopha n and serine, which produced poor substrates at the P-2 position, were amon g the best P-2' residues, The specificity at P-3 was generally opposite tha t of P-2. Residues at P-2, and to a lesser extent at P-3, influenced the cl eavage site. At the P-2 position, His, Phe, Tyr,Asn, or Trp produced cleava ge at the amino side of the first basic residue. In contrast, a P-2 Ile or Val produced cleavage between the dibasic pair, Other residues produced int ermediate effects, The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine, A comparison of the effect o f arginine or lysine at the P-1' or P-1 position showed that there is a ten dency to cleave on the amino side of arginine and that this cleavage produc es the highest k(cat) values.