Abstract
Continuous-flow mass spectrometry (CFMS) was used to monitor the products formed during the initial 0.25-20 s of the reactions catalyzed by the flavoprotein N-acetylpolyamine oxidase (PAO) and the pterin-dependent enzymes phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH). N,N'-Dibenzyl-1,4-diaminobutane (DBDB) is a substrate for PAO for which amine oxidation is rate-limiting. CFMS of the reaction showed formation of an initial imine due to oxidation of an exo-carbon-nitrogen bond. Nonenzymatic hydrolysis of the imine formed benzaldehyde and N-benzyl-1,4-diaminobutane; the subsequent oxidation by PAO of the latter to an additional imine could also be followed. Measurement of the deuterium kinetic isotope effect on DBDB oxidation by CFMS yielded a value of 7.6 ± 0.3, in good agreement with a value of 6.7 ± 0.6 from steady-state kinetic analyses. In the PheH reaction, the transient formation of the 4a-hydroxypterin product was readily detected; tandem mass spectrometry confirmed attachment of the oxygen to C(4a). With wild-type TyrH, the 4a-hydroxypterin was also the product. In contrast, no product other than a dihydropterin could be detected in the reaction of the mutant protein E332A TyrH.