Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase.
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 0.05-0.2 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.
Department of Biophysics, Escola Paulista de Medicina, São Paulo, Brazil.
Vitor
V
Oliveira
Reynaldo
R
Gatti
Vanessa
V
Rioli
Emer S
ES
Ferro
Alberto
A
Spisni
Antonio C M
AC
Camargo
Maria A
MA
Juliano
Luiz
L
Juliano
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D004795:Enzyme Stability; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology
D000596:Amino Acids; D008670:Metals; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012965:Sodium Chloride; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin