Supplementary Materialscb500848p_si_001. site reactivity SAHA will not depend on antibodies and allows perseverance of both poorly and highly reactive lysine residues therefore. In the band of 44 lysine residues with either no detectable prices or reactivity 30 10C5 MC1 sC1, just nine (or 12%) of the were discovered in at least among the natural data sets. On the other hand, 40 sites possess second order SAHA price constants 30 10C5 MC1 sC1 with 24 (or 29%) of these sites overlapping using the natural data models, and significantly 19/24 (or 79%) of these were seen in four different circumstances (Body ?(Figure3).3). For evaluation, we determined the next order rate constant of an unstructured histone H3 peptide with acetyl-CoA, yielding a value of 261 10C5 MC1 sC1 (Physique ?(Figure2).2). Lysine sites with the highest reactivity (second order rate constant) were found on ACAT1 and GDH, and many of these sites appear to dynamically change in biological conditions that compare SIRT3C/C to WT and caloric restriction (CR) to a control diet (CD; Figure ?Physique3).3). Together, these results Rabbit Polyclonal to FGFR1/2 suggest that many highly reactive sites are more likely to exhibit larger fold-changes between conditions and more likely to be targets of SIRT3. The extensive cataloguing of lysine reactivity allowed us to map these sites onto the protein structures of GDH and ACAT1, displaying a reactivity range that spans over 2 orders of magnitude. By visual inspection, lysine sites with the highest reactivity (red) tend to protrude away from the surface of the protein, while low reactivity sites (yellow) tend to form electrostatic interactions with neighboring residues (Figures ?(Figures22 and ?and4).4). In ACAT1, this point is usually illustrated by structurally comparing K84, which yielded no significant reactivity, with K260, K263, K265, and K270, which displayed rate constants ranging from 106 10C5 to 164 10C5 MC1 sC1. K84 is usually a part of a network of electrostatic interactions involving aspartate, glutamate, and arginine residues (Physique ?(Figure4A).4A). As a group, K260, K263, K265, and K270 form a cluster and do not make significant interactions with the protein surface (Physique ?(Figure4A).4A). Quite remarkably, the acetylation state of K260, K263, K265, and K270 increased 10-fold in the SIRT3C/C mice compare with WT (Physique ?(Figure33).4 Equally interesting is the observation that K260 and K265 acetylation decreases when comparing refed/fasted as well as obese/lean (Determine ?(Figure33).8 These residues are located in the CoA binding pocket, within 3C5 ? from the ribosyl-phosphate group of CoA. Site-specific acetyl-lysine incorporation and biochemical analysis provided direct evidence that SIRT3-mediated deacetylation of K260ac and K265ac enhanced ACAT1 activity, likely due to decreased affinity for coenzyme A (CoA) through lost electrostatic SAHA conversation between positively charged lysine and SAHA negatively charged 3-phosphate of CoA.8 Thus, the high intrinsic reactivity toward acetyl-CoA, described here, can identify functionally relevant acetylation sites, particularly those regulated by SIRT3. Open in a separate window Physique 4 Visualization of lysine reactivity. (A) Lysine reactivity mapped onto mouse ACAT1 structure (modeled from 2IB8, 87% identity; center panel). Reactivities of K260, K263, K265, K270 are shown in the acetyl-CoA binding pocket (left panel). non-reactive K84 shown developing a sodium bridge with E82 and D143 (correct -panel). (B) Lysine reactivity mapped onto bovine glutamate dehydrogenase (pdb: 3MW9; middle -panel). Reactivity of K503 proven close to the allosteric GTP binding site (still left panel). The trimeric antennae of GDH showing K480 and K477 reactivities and their close proximity. Acetylation price color scale is within Log10 space. We performed an identical structural evaluation of lysine residues from GDH, which is available being a homohexamer offering stacked.