Isomerization of Δ¹-Piperideine-2-Carboxylate to Δ²-Piperideine-2-Carboxylate on Complexation with Flavoprotein D-Amino Acid Oxidase

抄録

The 1, 646cm^-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1, 617cm^-1 upon 2^-13C substitu-tion of lysine. Thus, the band is assigned to a C(2)=C(3) stretching mode of the enamine, Δ²-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, Δ¹-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E+Im_??_EIM_??_EEn: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.