HEME-BIOSYNTHESIS IN MAMMALIAN SYSTEMS - EVIDENCE OF A SCHIFF-BASE LINKAGE BETWEEN THE PYRIDOXAL 5'-PHOSPHATE COFACTOR AND A LYSINE RESIDUEIN 5-AMINOLEVULINATE SYNTHASE

5-Aminolevulinate synthase is the first enzyme of the heme biosyntheti c pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevul inate synthase has been purified to homogeneity from an Escherichia co li overproducing strain, and the catalytic and spectroscopic propertie s of this recombinant enzyme were compared with those from nonrecombin ant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevufinate synthase is a pyridoxal 5'-phosphate-depen dent enzyme and is functional as a homodimer. The recombinant 5-aminol evulinate synthase holoenzyme was reduced with tritiated sodium borohy dride and digested with trypsin. A single peptide contained the majori ty of the label. The tritiated peptide was isolated, and its amino aci d sequence was determined; it corresponded to 15 amino acids around ly sine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-amin olevulinate synthase sequences and is present in the C-terminal (catal ytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, w hich is involved in the Schiff base linkage with pyridoxal 5'-phosphat e, from lysine to alanine or histidine abolished enzyme activity in th e expressed protein.