Defining the substrate specificity of mouse cathepsin P

被引:5
|
作者
Puzer, L
Barros, NMT
Oliveira, V
Juliano, MA
Lu, GZ
Hassanein, M
Juliano, L
Mason, RW
Carmona, AK
机构
[1] UNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 Sao Paulo, Brazil
[2] Univ Cidade Sao Paulo, Neurosci Lab, BR-03071000 Sao Paulo, Brazil
[3] Alfred I DuPont Hosp Children, Dept Biomed Res, Wilmington, DE 19803 USA
[4] Univ Delaware, Dept Biol Sci, Newark, DE 19716 USA
基金
巴西圣保罗研究基金会;
关键词
cysteine proteases; placentally expressed cathepsins; cathepsin P; flurogenic substrates; substrate specificity;
D O I
10.1016/j.abb.2004.12.007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitroplieiiyl]ethyleiiediamine). Systematic modifications were introduced resulting in five series of peptides to map the S-3 to S-2' subsites of the enzyme. The results indicate that the subsites S-1, S-2, S-1' and S-2', present a clear preference for hydrophobic residues. The specificity requirements of the S, subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. The S-3 subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). For several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na2SO4. No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P-3 to P-2' defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. The k(cat)/K-m for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:190 / 196
页数:7
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