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 pyridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPLC, the commercial PMP used in the incubation mixture was found to contain only extremely small amounts of PLP, which could not account for restoration of l-threonine dehydratase activity. HPLC analysis of the assay mixtures showed that during incubation, sufficient PLP had been formed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, which either contaminated, or were formed by the minimal amount of PLP-holoenzyme always present even in the dialyzed preparation. When sufficient PLP was formed, the PLP-holoenzyme and the original 'true' l-threonine dehydratase activity were restored. When PMP was incubated with the apoenzyme in the presence of small quantities of keto acids (pyruvate or 2-oxobutyrate) small amounts of l-alanine or l-aminobutyrate were formed. The reaction was not reversible; l-alanine and l-aminobutyrate did not react with the PLP-holoenzyme. No transaminating activity occurred with other amino acids. These results show that l-threonine dehydratase exists in two forms: the well known stable apoenzyme-PLP (hydrolase deaminating) and the transient apoenzyme-PMP (non-reversible half-transaminating). Half-transamination has the biological role of keeping the activity of the 'true' l-TDH constant and of regulating intracellular levels of pyruvate, alanine, oxobutyric acid, l-aminobutyric acid, l-threonine and l-serine.
Leoncini
R
R
Istituto di Biochimica e di Enzimologia, Pian dei Mantellini 44, 53100 Siena, Italy.
Vannoni
D
D
Di Pietro
M C
MC
Guerranti
R
R
Rosi
F
F
Pagani
R
R
Marinello
E
E
D000090:Acetoacetates / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D004793:Enzyme Reactivators / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013913:Threonine Dehydratase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000090:Acetoacetates; D001051:Apoenzymes; D004793:Enzyme Reactivators; C016635:acetoacetic acid; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D019289:Pyruvic Acid; D013913:Threonine Dehydratase; D000409:Alanine; C010627:pyridoxamine phosphate