Determination of the position of monooxygenation in the formation of catechol catalyzed by salicylate hydroxylase.

[3,5-2H]Salicylate was obtained by a hydrogen/deuterium exchange reaction carried out in 2H2O. Using this deuterated salicylate as a substrate for salicylate hydroxylase, the product catechol was isolated and characterized with respect to the positions of deuterium retention. Based on nuclear magnetic resonance and mass spectroscopy analyses, the product was identified as [3,5-2H]catechol. It is thus unequivocally demonstrated that salicylate is decarboxylated and hydroxylated at the same ring position. Also consistent with this conclusion is the lack of a kinetic isotope effect for the deuterated salicylate substrate.