Sirtuin chemical mechanisms

Abstract

Sirtuins are ancient proteins widely distributed in all lifeforms of earth. These proteins are universally able to bind NAD(+), and activate it to effect ADP-ribosylation of cellular nucleophiles. The most commonly observed sirtuin reaction is the ADP-ribosylation of acetyllysine, which leads to NAD(+)-dependent deacetylation. Other types of ADP-ribosylation have also been observed, including protein ADP-ribosylation, NAD(+) solvolysis and ADP-ribosyltransfer to 5,6-dimethylbenzimidazole, a reaction involved in eubacterial cobalamin biosynthesis. This review broadly surveys the chemistries and chemical mechanisms of these enzymes.

Figure 1

Depictions of fate of imidate complex partitioned between base exchange and deacetylation pathway. The formation of imidate is governed by the rate constant k₁, its reversal to the Michaelis complex is k₋₁ and k₂ is the rate of attack of the 2'-hydroxyl of the imidate. The two reaction coordinates are for enzymes that are sensitive to nicotinamide inhibition of deacetylation, and the bottom is proposed for the enzyme Af2Sir2, which is relatively insensitive to nicotinamide inhibition of deacetylation. The significance of these reaction coordinates is discussed in the text.

Scheme 1

Overall stoichiometry determined for the NAD⁺-dependent deacetylation reaction.

Scheme 2

Direct reaction of NAD⁺ with nucleophiles determined for distinct sirtuin enzymes. The top reaction depicts imidate and thioimidate formation. The second reaction depicts a direct solvolysis reaction characterized for a Plasmodium falciparum sirtuin enzyme. The bottom reaction represents an ADPribosyltransfer reaction determined for the CobB enzyme, a sirtuin from eubacteria.

Scheme 3

Proposed reaction mechanism of sirtuin catalyzed NAD⁺-dependent deacetylation. The asterisk depicts radioactively labeled nicotinamide that can be used to monitor the base exchange reaction (reversal of imidate to NAD⁺). Bottom scheme shows spontaneous non-enzymatic equilibration of AADPR isomers that occurs in solution.

Scheme 4

Different transition states or intermediates proposed for ADP-ribosylation of acetyllysine discussed in the text. Bond angles as drawn between nicotinamide and C1' and carbonyl-oxygen and C1' are not strictly defined in these representations.

Scheme 5

Methanolysis of imidate observed for a mutant and wildtype sirtuin. Sensitivity to nicotinamide inhibition is depicted by competitive attack of nicotinamide in base exchange.

Scheme 6

Mechanism of activation of sirtuin deacetylation reaction by isonicotinamide (INAM). INAM is able to compete with nicotinamide (NAM) to occupy position above b-face of imidate which pushes deacetylation reaction forward, and avoids NAM induced chemical reversal of the imidate, which is inhibitory to the deacetylation reaction.

Scheme 7

General mechanistic scheme that accounts for ADPribosyltransfers with inversion, retention and deacetylation as observed for Plasmodium falciparum Sir2 enzyme. In addition, nicotinamide reaction from imidate causes inhibitions of imidatesolvolysis and deacetylation. Nicotinamide does not inhibit the direct ADP-ribosyltransfer reaction shown by top arrow.