Coenzyme Models. 25. Facile Oxidation of Hemithiol Acetals by Flavin. Supporting Evidence for the Enediol Mechanism of Glyoxalase I

The rearrangement of hemithiol acetals (1) to -hydroxythiol esters (2) was inhibited almost completely on the addition of 3-methyltetra-O-acetylriboflavin (MeFl), and corresponding -keto acids were produced in good yields. Since (i) flavin is able to trap transient enediol and intermediates oxidatively and (ii) flavin does not serve as an efficient oxidant for intermediates including the 1,2-hydride shift mechanism (Cannizzaro reaction), the finding supports, in agreement with the results of Hall et al., the idea that the rearrangement (and also the enzymic catalysis by glyoxalase I) occurs via the enediol intermediate. The kinetic measurements, which were carried out conveniently by following the disappearance of the absorption band of MeFl, established that (i) the oxidation by MeFl is zero order in MeFl and first order in thiols and glyoxals, (ii) the rate constant for (dimethylamino)ethanethiol is greater by a factor of about 50 than that for 2-mercaptoethanol, (iii) the kH/kD values observed for C6H5COCHO vs. C6H5COCDO are 5-12, and (iv) the Kz value of the hemithiol acetal of methylglyoxal as carbon acid is estimated on the basis of linear free-energy relationships to be 10-11.3. The results show that the reaction sequence consists of the rate-limiting deprotonation from hemithiol acetal to form the enediol intermediate followed by the rapid oxidation by MeFl. It is proposed that the hemithiol acetal is classified as a carbon acid and the dissociation is facilitated owing to the electron-withdrawing nature of the acyl group and the carbanion-stabilizing effect of the sulfur atom.