Evidence of an essential lysine in pig liver 5-aminolevulinic acid dehydratase

Abstract

5-Aminolevulinic acid dehydratase catalyses the self condensation between two molecules of 5-aminolevulinic acid, via a Schiff base, in which a lysine residue at its active site is proposed to be involved. The aim of the work was to further clarify the mechanism of this step in porphobilinogen biosynthesis. 5-Aminolevulinic acid dehydratase was purified 230-fold from pig liver, ε -Aminolysil residues were identified by treating the enzyme with pyridoxal 5-phosphate. Schiff bases formed between either the substrate or pyridoxal 5-phosphate and the enzyme were stabilized by NaBH 4 reduction. Levulinate and pyruvate acted as competitive enzyme inhibitors. Pyridoxal 5-phosphate but not pyridoxamine 5-phosphate reversibly inhibited the enzyme activity, in a competitive fashion ( K i = 0.12 mM). After NaBH 4 treatment this inhibition became irreversible. The amount of labelled substrate bound to the enzyme after NaBH 4 reduction decreased in the presence of either pyridoxal 5-phosphate, levulinate or pyruvate. Enzyme elution profiles from Sephacryl S-300 showed that NaBH 4 treatment (1) in absence of substrate, did not induce any change on the enzyme, eluting as a typical 5-aminolevulinic acid dehydratase single peak (Mw 280,000), which overlapped with that of the enzyme activity; and (2) in the presence of labelled 5-aminolevulinate, had an additional peak eluting with a Mw of 140,000, without enzyme activity. This peak coincides in shape and elution volume with the radioactive one. These data suggest that pyruvate regulates pig liver 5-aminolevulinic acid dehydratase activity in vivo and that during catalysis, a tetrameric structure forms the enzyme-substrate complex. The results support the involvement of a critical ε -aminolysil group at the active site of the enzyme.