Aminolevulinate synthase: Lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis

Abstract

5‐Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine‐313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5′‐phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959–1965). Here we report on the effect of replacement of aminolevulinate synthase lysine‐313 by alanine, histidine, 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 indicate 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 co‐factor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase‐catalyzed reaction. In contrast, lysine‐313 is an essential catalytic residue, because the K313‐directed mutant enzymes have no measurable activity. In summary, site‐directed mutagenesis of the aminolevulinate synthase active‐site lysine‐313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5′‐phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine‐313 has a functional role in catalysis. Copyright © 2000 The Protein Society