Kinetics and comparative reactivity of human class I and class IIb histone deacetylases.
Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.
Schultz
Brian E
BE
Celera, 180 Kimball Way, South San Francisco, California 94080, USA. brian.schultz@celera.com
Misialek
Shawn
S
Wu
Jiansheng
J
Tang
Jie
J
Conn
Marion T
MT
Tahilramani
Ram
R
Wong
Lance
L
D001665:Binding Sites; D001667:Binding, Competitive; D003374:Coumarins / Q000737:chemistry; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D000073864:Histone Deacetylase 6; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000145:classification* / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000145:classification / Q000378:metabolism; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D000099186:Histone Deacetylase 3
D003374:Coumarins; D004791:Enzyme Inhibitors; D056284:Histone Deacetylase 1; D056464:Histone Deacetylase 2; D000073864:Histone Deacetylase 6; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D007527:Isoenzymes; D008239:Lysine; D011994:Recombinant Proteins; D012097:Repressor Proteins; D000099186:Histone Deacetylase 3; C012589:trichostatin A; C016949:N-epsilon-acetyllysine; C110550:HDAC1 protein, human; C447828:HDAC10 protein, human; C433811:HDAC6 protein, human; C409470:HDAC8 protein, human; C028743:7-amino-4-methylcoumarin