In order to obtain accurate information about the function of the ring-nitrogen of B6 in the enzymatic reactions dependent on the vitamin, the reaction rate leading to α-proton elimination from amino acid substrate was compared between native enzyme and holoenzyme species, recon-stituted with pyridoxal phosphate N-oxide (PLP N-oxide), using a stopped flow method. In tryptophanase, the formation rate of the deprotonated Schiff's base (A502nm complex) of the PLP N-oxide enzyme with L-alanine was about one-fifth of that of the native enzyme, being substan-tially consistent with the marked low catalytic activity of PL N-oxide in the corresponding nonenzymatic model reaction and of the PLP N-oxide enzyme in overall tryptophan degradation. This result stronglyy suggests that the electronegativity of the pyridinium-nitrogen plays a predominant role in the a-proton elimination from the substrate, one of the most important steps in overall reaction.
On the other hand, in aspartate aminotransferase (GOT) reaction, such a significant difference was not observed between the formation rate of deprotonated Schiff's base of native GOT with erythro-β-hydroxy-aspartate (A492nm, complex) and that of the artificial GOT activated with PLP N-oxide. However, the reaction of PMP N-oxide form of GOT with α-keto acid proceeded at an extremely slow rate as compared with that of PMP form of GOT. These phenomena indicate that the lower Vmax of overall GOT reaction catalyzed by the PLP N-oxide-bound holoenzyme would be accounted for by the marked smaller, reactivity of PMP N-oxide form with α-keto acid. These results strongly suggest that, in GOT, the α-proton elimination from substrate is mainly mediated by a nucleophilic attack of basic amino acid residue located at an ap-propriate position of enzyme protein and that the introduction of an oxygen atom into the pyridine-nitrogen of PLP results in a conforma-tional change of coenzyme analog-bound GOT which renders the interac-tion between PMP-form GOT with α-keto acid very difficult.
