Mechanisms of Formation and Equilibria of 4a and 5 Adducts of an Isoalloxazine. Reaction of 10-(2′,6′-Dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate with Sulfite in Aqueous Media

TY - JOUR

T1 - Mechanisms of Formation and Equilibria of 4a and 5 Adducts of an Isoalloxazine. Reaction of 10-(2′,6′-Dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate with Sulfite in Aqueous Media

AU - Bruice, Thomas C.

AU - Hevesi, Laszlo

AU - Shinkai, Seiji

PY - 1973/5/1

Y1 - 1973/5/1

N2 - The reaction of 10-(2′,6′-dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate (I) in aqueous sulfite-bisulfite buffers (30°, μ = 2.0) yields an equilibrium mixture of I plus 4a- and 5-sulfite adducts (4a, 5–, and 5H of eq 1). From the pH dependence of the relative concentration of 4a and the kinetically apparent acid dissociation constant of 5H (i.e., 5H → 5– with pKa5) the pH and buffer independent equilibrium constant (Ke = [5H]/[4a]) has been calculated to be 4.12 × 10–2. Therefore, at 30° in aqueous solution, the 4a adduct is thermodynamically favored over the neutral 5 adduct. From studies of the dependence of the equilibrium constants Kx = [4a]/[I] and Ky = [5–]/[I] upon pH and total sulfite buffer concentration ([S]T) it was determined that Kx was proportional to [HSO3 –]1.0 and Ky proportional to [SO3 2–]1.0. These dependencies of Kx and Ky upon concentrations of buffer species establish that the forward reactions from I to 4a and 5– have in their rate expressions the terms [HSO3 –] and [SO3 2–], respectively, in excess over the retrograde reactions of 4a → I and S– → I. The kinetics for approach to equilibrium in the conversion of I to products (4a, 5–, and 5H) were studied under the pseudo-first-order conditions of [buffer] ≫ [I0], The pseudo-first-order rate constants (kobSd) were found to be dependent upon three terms (eq 14); the first contained the product [HSO3 –][SO3 2–], the second [SO3 2–], and the third was independent of buffer species but dependent upon the mole fraction of a reactant of pKapp (pKa5) assignable to dissociation of 5H → 5–. With the knowledge of the dependence of the equilibrium ratios [4a]/[I] and [5–]/[I] upon [HSO3 –] and [SO3 2–], the rate terms were assignable (Scheme I) to: general acid (by HSO3 –) catalysis of nucleophilic attack of SO3 2– upon I to yield 4a and by microscopic reversibility general base (by SO3 2–) catalysis of conversion of 4a → I and unassisted nucleophilic attack of SO3 2– upon I to yield 5– with spontaneous conversion of 5– → I. An alternate scheme that would satisfy both the thermodynamic and kinetic findings would be that of Scheme II. Here I is converted to 5H via general acid (by HSO3 –) catalyzed attack of SO3 2– and to 5– by unassisted attack of SO3 2–, 4a arising from rearrangement of 5H. Arguments are presented which favor the mechanism of Scheme I.

AB - The reaction of 10-(2′,6′-dimethylphenyl)-3-methylisoalloxazine-6,8-disulfonate (I) in aqueous sulfite-bisulfite buffers (30°, μ = 2.0) yields an equilibrium mixture of I plus 4a- and 5-sulfite adducts (4a, 5–, and 5H of eq 1). From the pH dependence of the relative concentration of 4a and the kinetically apparent acid dissociation constant of 5H (i.e., 5H → 5– with pKa5) the pH and buffer independent equilibrium constant (Ke = [5H]/[4a]) has been calculated to be 4.12 × 10–2. Therefore, at 30° in aqueous solution, the 4a adduct is thermodynamically favored over the neutral 5 adduct. From studies of the dependence of the equilibrium constants Kx = [4a]/[I] and Ky = [5–]/[I] upon pH and total sulfite buffer concentration ([S]T) it was determined that Kx was proportional to [HSO3 –]1.0 and Ky proportional to [SO3 2–]1.0. These dependencies of Kx and Ky upon concentrations of buffer species establish that the forward reactions from I to 4a and 5– have in their rate expressions the terms [HSO3 –] and [SO3 2–], respectively, in excess over the retrograde reactions of 4a → I and S– → I. The kinetics for approach to equilibrium in the conversion of I to products (4a, 5–, and 5H) were studied under the pseudo-first-order conditions of [buffer] ≫ [I0], The pseudo-first-order rate constants (kobSd) were found to be dependent upon three terms (eq 14); the first contained the product [HSO3 –][SO3 2–], the second [SO3 2–], and the third was independent of buffer species but dependent upon the mole fraction of a reactant of pKapp (pKa5) assignable to dissociation of 5H → 5–. With the knowledge of the dependence of the equilibrium ratios [4a]/[I] and [5–]/[I] upon [HSO3 –] and [SO3 2–], the rate terms were assignable (Scheme I) to: general acid (by HSO3 –) catalysis of nucleophilic attack of SO3 2– upon I to yield 4a and by microscopic reversibility general base (by SO3 2–) catalysis of conversion of 4a → I and unassisted nucleophilic attack of SO3 2– upon I to yield 5– with spontaneous conversion of 5– → I. An alternate scheme that would satisfy both the thermodynamic and kinetic findings would be that of Scheme II. Here I is converted to 5H via general acid (by HSO3 –) catalyzed attack of SO3 2– and to 5– by unassisted attack of SO3 2–, 4a arising from rearrangement of 5H. Arguments are presented which favor the mechanism of Scheme I.

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U2 - 10.1021/bi00735a010

DO - 10.1021/bi00735a010

M3 - Article

VL - 12

SP - 2083

EP - 2089

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 11

ER -