Enzymatic elimination of fluoride from α-fluoro-β-alanine

Abstract Rat liver homogenates catalyzed the elimination of fluoride from ( R , S )- α -fluoro- β -alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial l -alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with l -alanine and glyoxylate as substrates, copurified with the α-fluoro-β-alanine-defluorinating enzyme. The NH 2 -terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of l -alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of k cat = 17 sec −1 , K L -Ala = 3.2 mM, and K glyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of ( R )- α -fluoro- β -alanine and ( S )- α -fluoro- β -alanine were k cat = 6.2 and 2.6 min −1 , respectively, and K m = 2.7 and 0.88 mM, respectively. l -Alanine potently inhibited the defluorination reaction with an apparent K i of 0.024 mM. ( R , S )- α -fluoro- β -alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridox-amino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with ( R , S )- α -fluoro- β -alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of ( R , S )- α -fluoro- β -alanine but was inactivated completely during dehalogenation of β-chloro- l -alanine.