期刊名称：Journal of Nutritional Science and Vitaminology
印刷版ISSN：0301-4800
电子版ISSN：1881-7742
出版年度：1973
卷号：19
期号：2
页码：129-143
DOI：10.3177/jnsv.19.129
出版社：Center for Academic Publications Japan
摘要：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 V max 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.
