Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Author Oliveira, Vitor Autor UNIFESP Google Scholar
Gatti, R Google Scholar
Rioli, V Google Scholar
Ferro, Emer Suavinho Autor UNIFESP Google Scholar
Spisni, A. Google Scholar
Camargo, Antonio Carlos Martins de Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Univ Parma
Inst Butantan
Abstract We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. the kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. the relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.054.2 M for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 M for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values; of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. the effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
Keywords protease
enthalpy of activation in proteolysis
entropy of activation in proteolysis
Language English
Date 2002-09-01
Published in European Journal of Biochemistry. Oxford: Blackwell Publishing Ltd, v. 269, n. 17, p. 4326-4334, 2002.
ISSN 0014-2956 (Sherpa/Romeo, impact factor)
Publisher Blackwell Publishing Ltd
Extent 4326-4334
Origin http://dx.doi.org/10.1046/j.1432-1033.2002.03129.x
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000177969100019
URI http://repositorio.unifesp.br/handle/11600/26958

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