A Continuous, Nonradioactive Assay for Histone Acetyltransferases 1 Youngjoo Kim, Kirk G. Tanner, and John M. Denu 2 Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098 Received December 6, 1999 Histone acetyltransferases (HATs) catalyze the acetyl-group transfer from acetyl-CoA to the -amino group of specific lysine residues within core histone proteins. HATs and other chromatin-remodeling en- zymes have been recently shown to regulate gene ac- tivation within specific loci. To facilitate mechanistic studies, we have developed two continuous, nonradio- active assays for the prototypical GCN5 HAT. The CoASH generated in the HAT reactions was continu- ously measured by using a coupled enzyme system with either -ketoglutarate dehydrogenase or pyru- vate dehydrogenase. The CoASH-dependent oxidation of -ketoglutarate or pyruvate is accompanied by the reduction of NAD to NADH, which was measured spec- trophotometrically 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 con- stants 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 as- say and compared. The 1.5- to 5-fold lower values ob- tained in the radioactive end-point assay are dis- cussed 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. © 2000 Academic Press Histone acetyltransferases (HATs) 3 are a family of enzymes which catalyze the transfer of an acetyl moi- ety from acetyl-CoA to the -amino group of lysine residues within core histone proteins. The acetylation of histones by HATs has an important role in transcrip- tional regulation by remodeling chromatin structure (1). In eukaryotes, approximately 146 base pairs of DNA wrap around an octamer of core histones to form the basic unit of chromatin, the nucleosome. This tightly packed DNA structure is transcriptionally re- pressive. HATs and other chromatin-remodeling en- zymes are proposed to generate an open, transcription- ally competent structure (2). Acetylation of histones neutralizes positively charged lysines in the amino- termini of histone proteins, thereby altering interac- tions between protein:protein and protein:DNA com- plexes. This loosening of chromatin structure allows access to transcription factors which initiate transcrip- tion of certain target genes (3). Several classes of HATs have been identified: p300/CBP (4), P/CAF (5), TAFII250 (6), and SRC-1 (7). Among HAT enzymes, yGCN5 (yeast GCN5), which is a P/CAF family mem- ber, has been the most characterized. The crystallo- graphic molecular model (8) and biochemical studies (9) of yGCN5 have suggested that a conserved acidic residue, Glu-173, acts as a general base by deprotonat- ing the -amino group of histone H3 substrate. The deprotonated -amino group then directly attacks the bound acetyl-CoA, resulting in transfer of an acetyl group to lysine. Except for a handful of biochemical studies, little is known concerning the mechanisms of catalysis, substrate specificity, and regulation. Under- standing the mechanism of HAT catalysis will provide deeper insight into the link between acetylation and gene activation. Thus, there is a great need for an 1 This work was supported by American Cancer Society Grant RPG-97-175-01-TBE, NIH Grant GM59785-01 and Postdoctoral fel- lowship (T32 DK07680) to K.G.T. 2 To whom correspondence should be addressed at Oregon Health Sciences University, Department of Biochemistry and Molecular Bi- ology, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. Fax: (503) 494-8393. E-mail: denuj@ohsu.edu. 3 Abbreviations used: HAT, histone acetyltransferases; Mes, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; TPP, thiamine pyrophosphate. 308 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 280, 308 –314 (2000) doi:10.1006/abio.2000.4546, available online at http://www.idealibrary.com on