Figure 6. Production of β-face Methanolysis Product.

(A) HPLC chromatograms showing the β-MeOADPR standard, and a Pf-Sir2 reaction run in the presence of H3 peptide and 30% MeOH. (B) Bottom: Pf-Sir2 catalyzed methanolysis with different concentrations of H3 Productions of α-MeOADPR (circle) and β-MeOADPR (square) with different concentrations of H3 are shown. The reactions contained 400 ∝M of NAD⁺ in 100 mM phosphate buffer with 0, 10, 25, 50, 75, 100, 150, 250, 400 and 500 ∝M of H3 all at pH 8.5. The following Michaelis parameters were determined by fitting the curves with KaleidaGraph: K_i (α-MeO-ADPR) = 70 ∝M, k₀ = 2.7 × 10⁻⁴ s⁻¹ K_m (β-MeO-ADPR) = 119 ∝M, k₅₀₀ = 1.6 × 10⁻⁴ s⁻¹ k_solvolysis ([H3] = 0) = 1.38 × 10⁻³ s⁻¹, k_solvolysis ([H3] = 500 ∝M) = 1.40 × 10⁻³ s⁻¹