| Phenylalanine aminomutase (PAM) derived from Taxus is a multifunctional enzyme that catalyzes the reversible vicinal exchange of the amino group and the pro-(3S) hydrogen of various ( 2S)-alpha-arylalanines to make the corresponding (3R)-beta-arylalanines, with trans-cinnamic acid as a major by-product. A combination of 1H- and 2H-NMR experiments were employed to analyze the stereochemistry at C2 of the beta-phenylalanine product catalyzed by PAM. [3,32H2]-(2S)-alpha-phenylalanine was incubated with PAM, and the biosynthetically-derived 2H-labeled beta-phenylalanine derivatized as the N-acetyl methyl ester, revealed deuterium chemical shift values consistent with those of [2,3-2H 2]-(2S,3R)-and (2R,3 S)-beta-phenylalanine racemate. Considering that chiral GC-MS analysis confirmed the beta-product to be the (3R)-enantiomer, and that an earlier study showed that the amino group migrates with retention of configuration at its terminus, the mutase was therefore shown to stereospecifically shuttle the pro-(3S) hydrogen to C2 with retention of configuration.;The deuterium kinetic isotope effect on Vmax/ KM is 2.0 +/- 0.2, indicating that Cbeta-H bond cleavage is rate limiting. The migratory deuterium of [2H 8]-(2S)-alpha-phenylalanine was shown to dynamically exchange (up to 70%) with protons from the solvent during the isomerization. The equilibrium constant of the enzyme for the natural substrate is 1.1 at 30°C.;The PAM reaction does not require an external cofactors but involves a post transcriptionally and autocatalytically synthesized 3,5-dihydro-5-methylidene- 4H-imidazolone (MIO) moiety which is believed to assist in shuttling the amino group between the alpha and beta positions of the substrate. The presence of the catalytic motif in PAM was confirmed by mutagenesis. Mutants S176A, S176G and S176T were used as blanks against wild-type enzyme in difference UV-visible spectroscopy with monitoring of the signature peak at 308 nm as observed in similar experiments with tyrosine aminomutase that also contain an MIO. The presence of the MIO in PAM was further confirmed by inhibition with cysteine at pH 10.5 under both aerobic and anaerobic conditions. An absorbance peak at 338 nm was observed which is typical of the formation of a cysteine-MIO complex as seen for the ammonia lyases. This absorbance maximum was absent when PAM mutants S176A, G and T were analyzed analogously. The PAM mutants (S176A, G T) converted 4'-nitro- and 4'-flourophenylalanines to their beta-isomers. Likely, the MIO, at least in part functions to reduce the pKa of the beta-hydrogens to facilitate the removal of the pro-3S hydrogen by a general base within the active site.;When E. coli transinfected with a plasmid carrying the pam gene is grown under light illumination it has a higher level of endogenously synthesized beta phenylalanine compared to non-illuminating conditions. |
