Kinetic mechanism of a novel HAT‐histone chaperone complex, Rtt109‐Vps75

Abstract

Rtt109 is a novel histone acetyltransferase (HAT) involved in promoting genomic stability, DNA repair and transcriptional regulation. A histone chaperone, either Vps75 or Asf1, is required for efficient acetyltransferase activity toward H3 histone substrates. In complex with Vps75, Rtt109 acetylates lysine 56 within the H3 globular domain, as well as N‐terminal tail residues on H3 peptide and H3‐H4 tetramer substrates. The ability of histone chaperones to activate and direct substrate targets is unique among HAT enzymes. Here we explore the molecular mechanism of histone acetylation and report a detailed kinetic investigation of the Rtt109‐Vps75 complex. A steady‐state kinetic analysis reveals evidence for a sequential kinetic mechanism whereby Rtt109‐Vps75 complex, acetyl‐CoA (Km= 0.27 μM) and H31–20 peptide substrates (Km=146 μM) form a complex prior to chemical catalysis (kcat =0.11 s−1). Product inhibition studies demonstrate that CoA competitively inhibits acetyl‐CoA, while thermodynamic binding measurements demonstrate that Rtt109‐Vps75, in the absence of H3 substrate, binds acetyl‐CoA and CoA with a Kd value of 1.41 μM and 43.29 μM, respectively. Additionally, pre‐steady state kinetic analysis suggests the chemical attack of substrate lysine on the bound acetyl‐CoA is the rate‐limiting step of catalysis. The pH profile of kcat reveals a crucial ionization of pKa=8.5, possibly representing the deprotonation of the substrate lysine on H3. Together, these data support a mechanism in which acetyl‐CoA binds first, and following H3 binding, the ε‐amino group of lysine directly attacks acetyl‐CoA, transferring the acetyl group to the targeted lysine residue. This research is funded by grants from the National Institutes of Health.