PLP Enzymes Research Articles

Binding of pyridoxal phosphate N-oxide to mitochondrial and cytoplasmic aspartate aminotransferases and some characteristics of the resulting artificial holoenzymes.

Specific features in the binding modes of pyridoxal phosphate N-oxide (PLP N-oxide) to mitochondrial and cytoplasmic aspartate transaminases (GOTm and GOTs) and some characteristics of the resulting artificial holoenzymes were studied. For the formation of catalytically active artificial holoenzymes, at least 10 min incubation was necessary. No significant changes were observed in the Michaelis-Menten constants of the substrates for the PLP N-oxide enzymes, except for a markedly large Km of aspartate for DOTS, as compared with those for the PLP enzymes. In general, the Km values of PLP N-oxide (Kco) were identical with those of PLP for both GOTm and DOTs, whereas the Vmax values of the enzymatic reactions catalyzed by the artificial holoenzymes de-creased to about one-half of those mediated by the native holoenzymes. In the case of GOTs, however, a complicated pattern was observed in the curve of the reaction rate vs. PLP N-oxide concentration, indicating a sort of negative cooperativity. Namely, the enzyme was activated in the presence of higher concentrations of PLP N-oxide. In this case, binding of 1 mole of PLP N-oxide to a certain lysine residue at a non-catalytic site was ascertained. The pH optimum of the PLP N-oxide-bound GOTs was 7.0 in K-phosphate buffer. On the other hand, the reaction of PLP N-oxide enzyme was greatly inhibited in Tris-HCl buffer (pH 7.5-9.0), and the PLP-enzyme showed a higher activity in this buffer.