| The mammalian sirtuins (SIRT1 -- 7) are NAD+-dependent protein deacetylases implicated in the regulation of diverse physiological processes such as insulin secretion, mitochondrial biogenesis, adipogenesis, DNA-damage responses, and cell survival. The involvement of sirtuins in these processes has stimulated an interest in studying them in biological contexts; however the methodologies to study these complex enzymes are limited.;Here, we describe several novel technological advances that enhance the study of the sirtuin family of enzymes. We have synthesized the novel tripeptide Nalpha-4-nitrobenzoyl-Nepsilon-acetyl-lysinyl-alaninyl-alaninyl-methyl ester (1) as a sirtuin substrate with K m values in the low micromolar range. We have used this substrate as a scaffold to synthesize the sirtuin inhibitor Nalpha-4-nitrobenzoyl-N epsilon-thioacetyl-lysinyl-alaninyl-alaninyl-methyl ester ( 2). With no known homology to any identified sirtuin substrate, we have shown that this simple scaffold is capable of inhibiting multiple members of the sirtuin family of enzymes with micromolar IC50 and nanomolar Ki values. In use with a modified NAD+ (3) we show that mechanistic trapping of sirtuins can be achieved via the formation of a temporally stable complex formed between 2, 3, and the enzyme can be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on strepavidin beads, followed by gel electrophoresis enabled detection and isolation of active sirtuins from the mixture.;We have used 1 as a scaffold for the synthesis of a tripeptide containing the novel lysine analog Nalpha-4-benzoyl-N epsilon-(acetamidooxy)lysine, (4). We present the groundwork for the development of a spectrophotometric sirtuin activity assay with the potential to measure the sirtuin-catalyzed deacetylation of 4 continuously without the use of an auxiliary enzyme.;We have also documented the hydrolytic instability of the commonly used sirtuin inhibitor sirtinol. Using NMR and UV/vis spectroscopy we have presented evidence of this compound's instability under aqueous conditions. |
