Solvent effects on thiamin-enzyme model interactions. I. Interactions with tryptophan

Abstract

The solvent polarity dependence of the interaction between thiamin and tryptophan was studied by spectrophotometric methods. The ultraviolet (UV) data clearly indicate that the interaction is weakened when the complex is transferred from water to aqueous ethanol or aqueous dioxane. The interaction of thiamin and tryptophan could also be detected by fluorescence-quenching studies (excitation of tryptophan at 287 nm, maximum emission at 348 nm). Appropriate treatment of the quenching data allowed dissection into static and dynamic contributions. A pyrimidine derivative related to thiamin, both in its neutral and protonated states, was shown to interact with tryptophan by fluorescence techniques, but not by UV. A thiazolium model was shown to interact with tryptophan by UV but was an inefficient quencher of the tryptophan fluorescence. Theoretical models are presented to explain the solvent dielectric constant dependence of the association constant between tryptophan and thiamin. Both electrostatic and dispersion forces are found to contribute to the stability of the complex.

Role of Glutamate 91 in Information Transfer during Substrate Activation of Yeast Pyruvate Decarboxylase

“…However, the sensitivity of the method depends on the number and location of the fluorophores. It had been reported that the binding of ThDP to the PDC apoenzyme quenches the intrinsic tryptophan fluorescence (34)(35)(36), suggesting that there is a tryptophan in the ThDP binding site (active site). The residue responsible for the source of the fluorescence quenching upon adding cofactors was identified as W487 in Zymomonas mobilis PDC (corresponding to W493 in yeast PDC) by site-directed mutagenesis (37).…”

Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-.ANG. resolution

“…4 from N4', orients its nitrogens to hydrogen bond with D28 and the carbonyl of W412 so that these potential hydrogen-bonding atoms do not make short contacts with N4'. It had been reported by several groups that reconstitution of apo-PDC (devoid of its cofactors) with ThDP quenches the intrinsic PDC fluorescence (Biaglow et al, 1969;Farzami et al, 1977;Jordan et al, 1988), suggesting interaction of coenzyme with a nearby tryptophan. In a recent study it was shown that the W493L mutant of PDC from Zymomonas mobilis is active (Diefenbach et al, 1992), but addition of ThDP leads to diminished fluorescence quenching compared to wild-type PDC.…”

N1'-Methylthiaminium diiodide. Model study on the effect of a coenzyme bound positive charge on reaction mechanisms requiring thiamin pyrophosphate

“…3 More recently the pyrimidine moiety was also shown to interact with tryptophan according to fluorescence experiments. 4 These results suggest potential binding mechanisms on the enzyme. Schellenberger and coworkers suggested that the amino group functions either as a proton acceptor (normal role for strongly basic amino groups) and/or in the transferral of acetaldehyde from coenzyme to solution.…”