Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates

Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates

Autor Johanning, K. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Lazure, C. Google Scholar
Lamango, N. S. Google Scholar
Steiner, D. F. Google Scholar
Lindberg, I Google Scholar
Instituição Louisiana State Univ
Universidade Federal de São Paulo (UNIFESP)
Inst Rech Clin Montreal
Univ Chicago
Resumo In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. in this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (k(cat)/K-m) between 9.4 x 10(4) M-1 s(-1)(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s(-1) (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s(-1) (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s(-1) (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited k(cat) of less than 0.05 s(-1). Substitution of ornithine for Lys at the P4 position did not significantly affect the k(cat) but increased the K-m 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1, These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters.
Idioma Inglês
Data de publicação 1998-08-28
Publicado em Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 273, n. 35, p. 22672-22680, 1998.
ISSN 0021-9258 (Sherpa/Romeo, fator de impacto)
Publicador Amer Soc Biochemistry Molecular Biology Inc
Extensão 22672-22680
Fonte http://dx.doi.org/10.1074/jbc.273.35.22672
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000075616600073
Endereço permanente http://repositorio.unifesp.br/handle/11600/25941

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