Restoration of rat liver L-threonine dehydratase activity by pyridoxamine 5P-phosphate: the half-transaminating activity of L-threonine dehydratase and its regulatory role Roberto Leoncini, Daniela Vannoni, M. Cristina Di Pietro, Roberto Guerranti, Francesca Rosi, Roberto Pagani, Enrico Marinello * Istituto di Biochimica e di Enzimologia, Pian dei Mantellini 44, 53100 Siena, Italy Received 14 April 1998; revised 30 July 1998; accepted 4 August 1998 Abstract 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 5P-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5P-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. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : L-Threonine dehydratase ; Pyridoxamine 5P-phosphate ; Pyridoxal 5P-phosphate ; Half-transamination 1. Introduction L-Threonine deaminase (L-TDH) is a pyridoxal 5P-phosphate (PLP)-dependent enzyme that catalyzes the decomposition of L-threonine and L-serine to 2-oxobutyrate and pyruvate, respectively, and am- monia. This reaction is referred to here as `true' de- aminase activity. The gluconeogenetic role and many properties of the enzyme are described in detail else- 0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII:S0304-4165(98)00094-4 Abbreviations : DTNB, 5,5P-dithiobis(2-nitrobenzoic acid); PCA, perchloric acid; PL, pyridoxal; PLP, pyridoxal 5P-phos- phate; PM, pyridoxamine; PMP, pyridoxamine 5P-phosphate ; SIM, standard incubation mixture ; L-TDH, L-threonine deami- nase * Corresponding author. Fax: +39 (577) 298057; E-mail : marinello@unisi.it Biochimica et Biophysica Acta 1425 (1998) 411^418