AMINOLEVULINATE SYNTHASE - LYSINE-313 IS NOT ESSENTIAL FOR BINDING THE PYRIDOXAL-PHOSPHATE COFACTOR BUT IS ESSENTIAL FOR CATALYSIS

5-Aminolevulinate synthase is the first enzyme of the heme biosyntheti c pathway in animals and some bacteria. Lysine-313 of the mouse erythr oid aminolevulinate synthase was recently identified to be linked cova lently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effec t of replacement of aminolevulinate synthase lysine-313 by alanine, hi stidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra i ndicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine s ubstrate is the first step in the mechanism of the aminolevulinate syn thase-catalyzed reaction. In contrast, lysine-313 is an essential cata lytic residue, because the K313-directed mutant enzymes have no measur able activity. In summary, site-directed mutagenesis of the aminolevul inate synthase active-site lysine-313, to alanine (K313A), histidine ( K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-p hosphate cofactor and the glycine substrate to produce external aldimi nes, but which are inactive. This suggests that lysine-313 has a funct ional role in catalysis.