The fluorinase catalyses an SN2-type nucleophilic substitution at the C-5' position of SAM, while L-methionine acts as a neutral leaving group. The fluorinase-catalysed reaction is estimated to be between 106 to 1015 times faster than the uncatalysed reaction, a significant rate enhancement. Despite this, the fluorinase is still regarded as a slow enzyme, with a turnover number of 0.06 min−1. The high kinetic barrier to reaction is attributed to the strong solvation of fluoride ion in water, resulting in a high activation energy associated with stripping solvating water molecules from aqueous fluoride ion, converting fluoride into a potent nucleophile within the active site. The reaction catalysed by the fluorinase is reversible, and upon incubation of 5'-fluoro-5'-deoxyadenosine and L-methionine with the fluorinase, SAM and fluoride ion are produced. Replacing L-methionine with L-selenomethionine results in a 6-fold rate enhancement of the reverse reaction, due to the increased nucleophilicity of the selenium centre compared to the sulfur centre. The fluorinase shows a degree of substrate tolerance for halide ion, and can also use chloride ion in place of fluoride ion. While the equilibrium for reaction between SAM and fluoride ion lies towards products FDA and L-methionine, the equilibrium position is reversed in the case for chloride ion. Incubation of SAM and chloride ion with the fluorinase does not result in generation of 5'-chloro-5'-deoxyadenosine, unless an additional enzyme, an L-amino acid oxidase, is added. The amino acid oxidase removes the L-methionine from the reaction, converting it to the corresponding oxo-acid. ion and the co-factor S-adenosyl-L-methioinine to generate 5'-chloro-5'-deoxyadenosine and L-methionine. The reaction only proceeds when L-methionine is removed from the reaction by an L-amino acid oxidase, driving the reaction equilibrium towards ClDA.The halide preference, coupled to the position of the two reaction equilibria allows for a nett transhalogenation reaction to be catalysed by the enzyme. Incubation of 5'-chloro nucleosides with the enzyme, along with catalytic L-selenomethionine or L-methionine results in the production of 5-fluoro nucleosides. When Fluorine-18|fluoride is used, this transhalogenation reaction can be used for the synthesis of radiotracers for positron emission tomography.
Structural studies
As of late 2007, 9 structures have been solved for this class of enzymes, with PDB accession codes,,,,,,,, and. The names given to the enzyme come not from the structure, but from the function: 5-Fluoro-5-deoxyadenosine is the molecule synthesised. The structure is homologous to the duf-62 enzyme series. The enzyme is a dimer of trimers. The active sites are located between these subunits , each can bind to one SAM molecule at a time.
