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. 2006 Jan 10;45(1):272-82.
doi: 10.1021/bi052014t.

Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine

Brian C Smith  1 , John M Denu
Affiliations

Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine

Brian C Smith et al. Biochemistry. .

Abstract

Sir2 NAD+-dependent protein deacetylases are implicated in a variety of cellular processes such as apoptosis, gene silencing, life-span regulation, and fatty acid metabolism. Despite this, there have been relatively few investigations into the detailed chemical mechanism. Sir2 proteins (sirtuins) catalyze the chemical conversion of NAD+ and acetylated lysine to nicotinamide, deacetylated lysine, and 2'-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18O label from the peptide acetyl group to the ribose 1'-position of OAADPr, providing direct evidence for the formation of a covalent alpha-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the alpha-1'-O-alkylamidate intermediate to yield beta-1'-O-methyl-ADP-ribose in a Sir2 histidine-to-alanine mutant. This conserved histidine (His-135 in HST2) activates the ribose 2'-hydroxyl for attack on the alpha-1'-O-alkylamidate. The histidine mutant is stalled at the intermediate, allowing water and other alcohols to compete kinetically with the attacking 2'-hydroxyl. Measurement of the pH dependence of kcat and kcat/Km values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD+ binding and in general-base activation of the 2'-hydroxyl. Also, transfer of an 18O label from water to the carbonyl oxygen of the acetyl group in OAADPr is consistent with water addition to the proposed 1',2'-cyclic intermediate formed after 2'-hydroxyl attack on the alpha-1'-O-alkylamidate. The effect of pH and of solvent viscosity on the kcat values suggests that final product release is rate-limiting in the wild-type enzyme. Implications of this new evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.

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Figures

Scheme 1
Scheme 1
General reaction catalyzed by Sir2 protein deacetylases.
Scheme 2
Scheme 2
Proposed mechanism of formation of both ADPr and OAADPr in HST2 H135A.
Figure 1
Figure 1
(A) 18O-labelling experiments with HST2 WT. Reactions contained 1 mM DTT, 200 μM18O-AcH3, 185 μM NAD+, 10 μM HST2, in natural abundance water or 90% 18OH2, and 20 mM pyridine buffer at pH 7. Each reaction was split into three aliquots, two of which were lyophilized and exchanged in 10% formic acid and 90% natural abundance OH2 or 18OH2. 18O incorporation was determined by examining the relative peak heights at ∼600.5, ∼602.5, and ∼604.5 m/z corresponding to zero, one, or two 18O atoms. (B) The rate of non-enzymatic exchange of an 18O-label from water into OAADPr. Purified OAADPr was diluted to 16.5 μM in 20 mM pyridine buffer and 90% 18OH2 at pH 7. Timepoints were injected directly into the mass spectrometer. The y-intercept, which represents the zero-timepoint before exposing OAADPr to 18O-water, was calculated as 13.3%, and is consistent with the natural distribution of heavy isotopes across the entire OAADPr molecule. A rate of non-enzymatic incorporation of 0.15% per minute was calculated, which corresponds to a maximum of 4.5% non-enzymatic incorporation for the 30-minute reaction time used in our assays.
Figure 1
Figure 1
(A) 18O-labelling experiments with HST2 WT. Reactions contained 1 mM DTT, 200 μM18O-AcH3, 185 μM NAD+, 10 μM HST2, in natural abundance water or 90% 18OH2, and 20 mM pyridine buffer at pH 7. Each reaction was split into three aliquots, two of which were lyophilized and exchanged in 10% formic acid and 90% natural abundance OH2 or 18OH2. 18O incorporation was determined by examining the relative peak heights at ∼600.5, ∼602.5, and ∼604.5 m/z corresponding to zero, one, or two 18O atoms. (B) The rate of non-enzymatic exchange of an 18O-label from water into OAADPr. Purified OAADPr was diluted to 16.5 μM in 20 mM pyridine buffer and 90% 18OH2 at pH 7. Timepoints were injected directly into the mass spectrometer. The y-intercept, which represents the zero-timepoint before exposing OAADPr to 18O-water, was calculated as 13.3%, and is consistent with the natural distribution of heavy isotopes across the entire OAADPr molecule. A rate of non-enzymatic incorporation of 0.15% per minute was calculated, which corresponds to a maximum of 4.5% non-enzymatic incorporation for the 30-minute reaction time used in our assays.
Figure 2
Figure 2
pH rate profiles. Reaction rates were determined based on OAADPr formation. Reactions for HST2 WT contained 150 μM 3H-AcH3, 1 mM DTT, 0.02−2 μM HST2 WT, and varying concentrations of NAD+ from 5−320 μM in TBA buffer. Reaction for HST2 H135A contained 2−3 mM 3H-AcH3, 1 mM DTT, 2−5 μM HST2 H135A, and varying concentrations of NAD+ from 5−1280 μM in TBA buffer. (A) Effect of pH on kcat/Km varying NAD+ for HST2 WT (squares) and HST2 H135A (circles). HST2 WT displayed a critical ionization with a pKa of 6.77 ± 0.06, while HST2 H135A exhibited no effect over the pH range tested. (B) Effect of pH on kcat for HST2 WT (squares) and HST2 H135A (circles). HST2 H135A displayed a critical ionization with a pKa of 7.43 ± 0.13 while HST2 WT showed no critical ionizations over the pH range tested.
Figure 2
Figure 2
pH rate profiles. Reaction rates were determined based on OAADPr formation. Reactions for HST2 WT contained 150 μM 3H-AcH3, 1 mM DTT, 0.02−2 μM HST2 WT, and varying concentrations of NAD+ from 5−320 μM in TBA buffer. Reaction for HST2 H135A contained 2−3 mM 3H-AcH3, 1 mM DTT, 2−5 μM HST2 H135A, and varying concentrations of NAD+ from 5−1280 μM in TBA buffer. (A) Effect of pH on kcat/Km varying NAD+ for HST2 WT (squares) and HST2 H135A (circles). HST2 WT displayed a critical ionization with a pKa of 6.77 ± 0.06, while HST2 H135A exhibited no effect over the pH range tested. (B) Effect of pH on kcat for HST2 WT (squares) and HST2 H135A (circles). HST2 H135A displayed a critical ionization with a pKa of 7.43 ± 0.13 while HST2 WT showed no critical ionizations over the pH range tested.
Figure 3
Figure 3
Effect of viscosity on kcat. Reaction rates were determined based on OAADPr formation. kcat0/kcat is the kcat the absence of sucrose divided by the kcat measured at each concentration of sucrose. HST2 WT (squares) reactions contained 500 μM NAD+, 2−64 μM AcH3 (11-mer) from, 1 mM DTT, 0−34% w/v sucrose, and 30−40 nM HST2 WT in TBA buffer at pH 8. HST2 H135A (circles) reactions contained 2 mM NAD+, 150−2400 μM AcH3 (11-mer), 1 mM DTT, 0−34% w/v sucrose, and 30−40 nM HST2 WT in TBA buffer at pH 8. Linear regression analysis showed a slope of 1.15 ± 0.07 for HST2 WT and 0.02 ± 0.12 for HST2 H135A.
Figure 4
Figure 4
The relative formation of ADPr and OAADPr. Reactions contained 400−600 μM AcH3 (11-mer), 1 mM DTT, 500−1000 μM 32P-NAD+, 20−200 μM HST2 H135A or 0.5−50 μM HST2 WT, and 20−50 mM Tris-Cl buffer at pH 7.5 and 25 °C. The ADPr observed from HST2 WT was at background levels and its formation did not increase over time. Each column was normalized to the total product formed a each timepoint. Error bars represent standard deviations.
Figure 5
Figure 5
18O-labelling experiments with HST2 H135A. Reactions contained 1 mM DTT, 400 μM AcH3 (11-mer), 500 μM NAD+, 50 μM HST2 H135A, 84% 18OH2, and 20 mM pyridine buffer adjusted at pH 7 were reacted for 30 minutes at 25 °C. Reactions were split into three aliquots, two of which were lyophilized and then exchanged in 10% formic acid and 90% natural abundance OH2 or 18OH2. 18O incorporation was determined by examining the relative peak heights at ∼600.5, ∼602.5, and ∼604.5 m/z corresponding to zero, one, or two 18O atoms.
Figure 6
Figure 6
(A) ESI-MS of the 30 minute reaction of HST2 H135A with 1 mM DTT, 400 μM AcH3 (11-mer), 500 μM NAD+, 50 μM HST2 H135A, 20% v/v MeOH, and 20 mM pyridine buffer at pH 7 at 25 °C. HST2 H135A forms ADPr, 1’-O-methyl-ADPr, and OAADPr under these conditions. (B) ESI-MS of the 30 minute control reaction of HST2 WT with 1 mM DTT, 400 μM AcH3 (11-mer), 500 μM NAD+, 21 μM HST2 WT, 20% v/v MeOH, and 20 mM pyridine buffer at pH 7 at 25 °C. HST2 WT forms no detectable 1’-O-methyl-ADPr under these reaction conditions. (C) ESI-MS of the 30 minute non-enzymatic incorporation of 20% v/v MeOH into 500 μM ADPr with 1 mM DTT, 400 μM AcH3 (11-mer), and 20 mM pyridine buffer at pH 7 at 25 °C. There was no detectable 1’-O-methyl-ADPr in this control. Therefore, the 1’-O-methyl-ADPr observed with HST2 H135A was due to enzymatic activity inherent in the mutant enzyme only.
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