Studies on the Subsite Specificity of Rat Nardilysin (N-Arginine Dibasic Convertase)

• Published in: The Journal of biological chemistry Vol. 275; no. 26; pp. 19545 - 19551
• Main Authors: Chow, K.Martin, Csuhai, Eva, Juliano, Maria Aparecida, St. Pyrek, Jan, Juliano, Luiz, Hersh, Louis B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30-06-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acids - metabolism; Animals; Binding Sites; Chromatography, High Pressure Liquid; Fluorometry; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Metalloendopeptidases - chemistry; Metalloendopeptidases - metabolism; Peptides - metabolism; Rats; Substrate Specificity; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M909020199

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX1X2RKX3GQ-ethylenediamine-2,4-dinitrophenyl, 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 onKm among different residues at any of these positions. In contrast, residues at each position affectedkcat, 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 P2position, 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 highestkcat values.