On the basis of MD simulations, we synthesized LANA-DBM-BAHA catalysts 21-25, in which BAHA was conjugated with a Lys residue introduced at either of G1-5 positions (Figure ). As negative control catalysts, we synthesized 26 and 27, which lack DBM and acidic patch-binding ability, respectively (Figure ). Remarkably, regioselectivity of the catalysts dramatically changed depending on the position of BAHA conjugation. Consistent with MD simulations, 21 (BAHA at G1) promoted acetylation at H2BK43/46 and H2BK120 (Figure ). Yield of H2BK120ac was higher than that of H2BK43/46ac, probably due to the higher intrinsic reactivity of H2BK120 (Figure ). In contrast, 25 (BAHA at G5) promoted acetylation mainly at H2BK43 (25% yield) and slightly at H2BK46 (5% yield) (Figure ). H2BK120 was rarely acetylated by 25, as H2BK120 was outside CER (Figure ). 26 and 27 did not promote H2BK43-selective acetylation efficiently (Figure ), indicating that bivalent nucleosome binding of the catalyst was essential. Comprehensive quantification of Lys acetylation yield within histones, promoted by 25, showed that other Lys residues than H2BK43 or H2BK46 were less acetylated (Figure ). Since both H2BK43 and H2BK46 were within CER of 25 (Figure ) and intrinsic reactivities of H2BK43 and H2BK46 were comparable (Figure ), the significantly higher yield of H2BK43 than H2BK46 could not be explained only by the distance and intrinsic reactivity parameters. We propose the conformational flexibility (i.e., fluctuation) of Lys residues as the third determinant of yield and regioselectivity. The lower flexibility of H2BK43 than H2BK46 (Table ) would contribute to the higher rate of the catalytic histone acylation reaction.