A continuous, nonradioactive assay for histone acetyltransferases.
Histone acetyltransferases (HATs) catalyze the acetyl-group transfer from acetyl-CoA to the epsilon-amino group of specific lysine residues within core histone proteins. HATs and other chromatin-remodeling enzymes have been recently shown to regulate gene activation within specific loci. To facilitate mechanistic studies, we have developed two continuous, nonradioactive assays for the prototypical GCN5 HAT. The CoASH generated in the HAT reactions was continuously measured by using a coupled enzyme system with either alpha-ketoglutarate dehydrogenase or pyruvate dehydrogenase. The CoASH-dependent oxidation of alpha-ketoglutarate or pyruvate is accompanied by the reduction of NAD to NADH, which was measured spectrophotometrically at 340 nm. The steady-state rate constants with substrates acetyl-CoA and a synthetic peptide (corresponding to the first 20 amino acids of H3 histone) were determined. The resulting rate constants were not significantly different between the two coupled assays, providing strong validation of these methods. Rate constants were also determined using the commonly employed radioactive filter-binding assay and compared. The 1.5- to 5-fold lower values obtained in the radioactive end-point assay are discussed in terms of the technical problems and limitations of this assay. The coupled assays should be widely applicable since the production of CoASH is common to all HAT enzymes, regardless of protein substrate.
Kim
Y
Y
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon, 97201-3098, USA.
Tanner
K G
KG
Denu
J M
JM
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000032:analysis*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000032:analysis*; D051548:Histone Acetyltransferases; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry / Q000379:methods*; D015003:Yeasts; D000099224:p300-CBP-Associated Factor
D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051548:Histone Acetyltransferases; D006657:Histones; D011494:Protein Kinases; D029701:Saccharomyces cerevisiae Proteins; C501392:GCN5 protein, S cerevisiae; D000099224:p300-CBP-Associated Factor