Effect of lysine side chain length on histone lysine acetyltransferase catalysis

Abstract

Histone lysine acetyltransferase (KAT)-catalyzed acetylation of lysine residues in histone tails plays a key role in regulating gene expression in eukaryotes. Here, we examined the role of lysine side chain length in the catalytic activity of human KATs by incorporating shorter and longer lysine analogs into synthetic histone H3 and H4 peptides. The enzymatic activity of MOF, PCAF and GCN5 acetyltransferases towards histone peptides bearing lysine analogs was evaluated using MALDI-TOF MS assays. Our results demonstrate that human KAT enzymes have an ability to catalyze an efficient acetylation of longer lysine analogs, whereas shorter lysine analogs are not substrates for KATs. Kinetics analyses showed that lysine is a superior KAT substrate to its analogs with altered chain length, implying that lysine has an optimal chain length for KAT-catalyzed acetylation reaction.

Substrate Scope for Human Histone Lysine Acetyltransferase KAT8

“…The incubation of an equimolar amount of H4K16 and inhibitor peptide H4K*16 (100 µM) with KAT8 revealed that none of the histone peptides exhibited significant inhibition potency ( Figure S24 ). This result is in line with previous findings, most likely dictated by the low binding affinity of histone peptides towards KAT8 [ 10 , 29 ].…”

“…Controls in the absence of KAT8 were carried out in parallel to demonstrate the enzymatic nature of the acetyl transfer reactions. KAT8-catalysed acetylation of H4K16 was quantitative after 1 h, in line with previous findings ( Figure 3 a and Figure S17 ) [ 10 , 11 ]. We examined the substrate specificity of the natural lysine analogue Kme, observing that it did not undergo KAT8-catalysed acetylation ( Figure 3 b), even after prolonged incubation and higher enzyme concentration (6 h; 10 µM KAT8) ( Figure S18a ), indicating that sterics on N ε play an important role in efficient KAT8 catalysis.…”

“…The biocatalytic potential of KAT8 was assessed through a MALDI-TOF MS based enzymatic assay [ 10 , 11 ]. Human KAT8 was incubated in the presence of H4K*16 peptides and AcCoA under “standard conditions” (2 µM KAT8, 100 µM histone peptide, 300 µM AcCoA; assay buffer: 50 mM Hepes, 0.1 mM EDTA, 1 mM DTT, pH = 8.0) and the production of acetylated peptides was monitored at different time points.…”

“…On the other hand, none of the sterically demanding lysine analogues, F 4a , F 3a and F 3me-a , underwent acetylation by KAT8 within the limit of detection under standard conditions ( Figure 3 j–l), or optimised conditions (6 h, 10 µM KAT8) ( Figure S18f–h ). Interestingly, the result seems to not be solely dependent on the decreased nucleophilicity of the aniline-containing analogues (F 4a , F 3a ), due to the fact that the alkyl aromatic amine-containing peptide (F 3me-a ) also did not undergo KAT8 acetylation, even though it is structurally related to homolysine (hK), an example of a good KAT8 substrate [ 10 ]. The results are in line with recent findings on human KMTs, showing a lack of methylation of bulkier lysine analogues [ 24 ].…”

“…Nevertheless, in all KATs, the deprotonation of the lysine N ε -amino group is achieved by highly conserved glutamic or aspartic acid in the active site [ 8 ]. In addition to KATs’ renowned ability of transferring bulkier acyl groups from their respective acyl-CoA co-substrates [ 9 ], recent investigations shed light on the ability of KATs to catalyse the acetylation of lysine analogues with a longer chain length [ 10 ], and the acylation of γ-thialysine, a simple lysine analogue site-specifically introduced on histone tails [ 11 ]. Despite the recent striking progress achieved in the field of epigenetic drug discovery, due to the Food and Drug Administration (FDA) approval of several KDAC inhibitors for the treatment of different cancers and numerous clinical candidates targeting bromodomains, we are currently lacking potent and specific inhibitors or modulators of KAT activity [ 12 , 13 ].…”