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@article{1640657,
author = {Zessin, M. and Kutil, Z. and Meleshin, M. and Novakova, Z. and Ghazy, E. and Kalbas, D. and Marek, Martin and Romier, C. and Sippl, W. and Barinka, C. and Schutkowski, M.},
article_location = {Washington},
article_number = {48},
doi = {http://dx.doi.org/10.1021/acs.biochem.9b00786},
keywords = {FLUORESCENCE QUENCHING PROBES; EPSILON-THIOACETYL-LYSINE; DEACETYLASE ASSAY; SIR2 FAMILY; POSTTRANSLATIONAL MODIFICATIONS; PEPTIDE ARRAYS; DECROTONYLASE ACTIVITY; SUBSTRATE-SPECIFICITY; THIOAMIDE SUBSTRATE; CATALYTIC-ACTIVITY},
language = {eng},
issn = {0006-2960},
journal = {Biochemistry},
title = {One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity},
url = {https://pubs.acs.org/doi/10.1021/acs.biochem.9b00786},
volume = {58},
year = {2019}
}
TY - JOUR
ID - 1640657
AU - Zessin, M. - Kutil, Z. - Meleshin, M. - Novakova, Z. - Ghazy, E. - Kalbas, D. - Marek, Martin - Romier, C. - Sippl, W. - Barinka, C. - Schutkowski, M.
PY - 2019
TI - One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity
JF - Biochemistry
VL - 58
IS - 48
SP - 4777-4789
EP - 4777-4789
PB - American Chemical Society
SN - 00062960
KW - FLUORESCENCE QUENCHING PROBES
KW - EPSILON-THIOACETYL-LYSINE
KW - DEACETYLASE ASSAY
KW - SIR2 FAMILY
KW - POSTTRANSLATIONAL MODIFICATIONS
KW - PEPTIDE ARRAYS
KW - DECROTONYLASE ACTIVITY
KW - SUBSTRATE-SPECIFICITY
KW - THIOAMIDE SUBSTRATE
KW - CATALYTIC-ACTIVITY
UR - https://pubs.acs.org/doi/10.1021/acs.biochem.9b00786
L2 - https://pubs.acs.org/doi/10.1021/acs.biochem.9b00786
N2 - We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced k(cat) values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an similar to 5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s(-1), which are more than 100-fold more effective than most of the known su bstrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 mu M for an N-methylated, N-myristoylated peptide derivative and human HDAC11.
ER -
ZESSIN, M., Z. KUTIL, M. MELESHIN, Z. NOVAKOVA, E. GHAZY, D. KALBAS, Martin MAREK, C. ROMIER, W. SIPPL, C. BARINKA a M. SCHUTKOWSKI. One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity. \textit{Biochemistry}. Washington: American Chemical Society, 2019, roč.~58, č.~48, s.~4777-4789. ISSN~0006-2960. Dostupné z: https://dx.doi.org/10.1021/acs.biochem.9b00786.
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