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.
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
Udayanga
U
Wanninayake
Kevin D
KD
Walker
D000642:Ammonia-Lyases / Q000378:metabolism*; D002934:Cinnamates / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D020636:Pantoea / Q000201:enzymology; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D020946:Taxus / Q000201:enzymology
D002934:Cinnamates; D007093:Imidazoles; C117197:imidazolone; D000642:Ammonia-Lyases; D010650:Phenylalanine Ammonia-Lyase