Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin

Substrate specificity characterization of recombinant metallo oligo-peptidases thimet oligopeptidase and neurolysin

Author Oliveira, V Google Scholar
Campos, M. Google Scholar
Melo, R. L. Google Scholar
Ferro, E. S. Google Scholar
Camargo, ACM Google Scholar
Juliano, M. A. Google Scholar
Juliano, L. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Universidade de São Paulo (USP)
Inst Butantan
Abstract We report a systematic and detailed analysis of recombinant neurolysin (EC 3.4.24.16) specificity in parallel with thimet oligopeptidase (TOP, EC 3.4.24.15) using Bk sequence and its C- and N-terminal extensions as in human kininogen as motif for synthesis of internally quenched fluorescent substrates, the influence of the substrate size was investigated, and the longest peptide susceptible to TOP and neurolysin contains 17 amino acids. the specificities of both oligopeptidases to substrate sites P(4) to P(3)' were also characterized in great detail using seven series of peptides based on Abz-GFSPFRQ-EDDnp taken as reference substrate. Most of the peptides were hydrolyzed at the bond corresponding to P(4)-F(5) in the reference substrate and some of them were hydrolyzed at this bond or at F(2)-S(3) bond. No restricted specificity was found for P(1)' as found in thermolysin as well for P(1) substrate position, however the modifications at this position (P(1)) showed to have large influence on the catalytic constant and the best substrates for TOP contained at P(1), Phe, Ala, or Arg and for neurolysin Asn or Arg. Some amino acid residues have large influence on the K(m) constants independently of its position. On the basis of these results, we are hypothesizing that some amino acids of the substrates can bind to different sub-sites of the enzyme fitting P-F or F-S bond, which requires rapid interchange for the different forms of interaction and convenient conformations of the substrate in order to expose and fit the cleavage bonds in correct position for an efficient hydrolysis. Finally, this plasticity of interaction with the substrates can be an essential property for a class of cytosolic oligopeptidases that are candidates to participate in the selection of the peptides to be presented by the MHC class I.
Language English
Date 2001-04-10
Published in Biochemistry. Washington: Amer Chemical Soc, v. 40, n. 14, p. 4417-4425, 2001.
ISSN 0006-2960 (Sherpa/Romeo, impact factor)
Publisher Amer Chemical Soc
Extent 4417-4425
Origin http://dx.doi.org/10.1021/bi002715k
Access rights Closed access
Type Article
Web of Science ID WOS:000168198400025
URI http://repositorio.unifesp.br/handle/11600/26529

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