Assessing the deamination rate of a covalent aminomutase adduct by burst phase analysis

Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated-methylidene imidazolone (NH(2)-MIO) adduct of a Taxus phenylalanine aminomutase. The kinetic parameters were interrogated by a non-natural substrate (S)-styryl-α-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH(2)-MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model demonstrating that the NH(2)-MIO adduct and cinnamate intermediate are sufficiently retained to catalyze the natural α- to β-phenylalanine isomerization.