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

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2000-06-30
Autores
Chow, K. M.
Csuhai, E.
Juliano, Maria Aparecida [UNIFESP]
St Pyrek, J.
Juliano, Luiz [UNIFESP]
Hersh, L. B.
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The subsite specificity of rat nardilysin was investigated using fluorogenic 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 nomenclature of Schechter and Berger (Schechter, L, and Berger, A. (1967) Biochem, 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 designated P-2, P-3, P-2', P-3', etc.) There was little effect on II, among different residues at any of these positions, in contrast, residues at each position 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, Tryptophan and serine, which produced poor substrates at the P-2 position, were among the best P-2' residues, the specificity at P-3 was generally opposite that of P-2. Residues at P-2, and to a lesser extent at P-3, influenced the cleavage site. At the P-2 position, His, Phe, Tyr,Asn, or Trp produced cleavage 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 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 P-1' or P-1 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.
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Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 275, n. 26, p. 19545-19551, 2000.
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