کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9882321 | 1536553 | 2005 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Defining the substrate specificity of mouse cathepsin P
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
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-dinitrophenyl]ethylenediamine). Systematic modifications were introduced resulting in five series of peptides to map the S3 to S2â² subsites of the enzyme. The results indicate that the subsites S1, S2, S1â², and S2â², present a clear preference for hydrophobic residues. The specificity requirements of the S2 subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. The S3 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 P3 to P2â² 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 kcat/Km 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 435, Issue 1, 1 March 2005, Pages 190-196
Journal: Archives of Biochemistry and Biophysics - Volume 435, Issue 1, 1 March 2005, Pages 190-196
نویسندگان
Luciano Puzer, Nilana M.T. Barros, Vitor Oliveira, Maria Aparecida Juliano, Guizhen Lu, Mohamed Hassanein, Luiz Juliano, Robert W. Mason, Adriana K. Carmona,