RESTORATION OF RAT-LIVER L-THREONINE DEHYDRATASE ACTIVITY BY PYRIDOXAMINE 5'-PHOSPHATE - THE HALF-TRANSAMINATING ACTIVITY OF L-THREONINE DEHYDRATASE AND ITS REGULATORY ROLE

When a highly purified preparation of rat liver L-threonine deaminase (L-TDH, EC 4.2.1.16) was 99% inactivated by dialysis, removing bound p yridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPL C, the commercial PMP used in the incubation mixture was found to cont ain only extremely small amounts of PLP, which could not account for r estoration of L-threonine dehydratase activity. HPLC analysis of the a ssay mixtures showed that during incubation, sufficient PLP had been f ormed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, whic h either contaminated, or were formed by the minimal amount of PLP-hol oenzyme always present even in the dialyzed preparation. When sufficie nt PLP was formed, the PLP-holoenzyme and the original 'true' L-threon ine dehydratase activity were restored. When PMP was incubated with th e apoenzyme in the presence of small quantities of keto acids (pyruvat e or 2-oxobutyrate) small amounts of L-alanine or L-aminobutyrate were formed. The reaction was not reversible; L-alanine and L-aminobutyrat e did not react with the PLP-holoenzyme. No transaminating activity oc curred with other amino acids. These results show that L-threonine deh ydratase exists in two forms: the well known stable apoenzyme-PLP (hyd rolase deaminating) and the transient apoenzyme-PMP (non-reversible ha lf-transaminating). Half-transamination has the biological role of kee ping the activity of the 'true' L-TDH constant and of regulating intra cellular levels of pyruvate, alanine, oxobutyric acid, L-aminobutyric acid, L-threonine and L-serine. (C) 1998 Elsevier Science B.V. All rig hts reserved.