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 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 cofactor 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.
Ferreira
G C
GC
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612, USA.
Vajapey
U
U
Hafez
O
O
Hunter
G A
GA
Barber
M J
MJ
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D000624:5-Aminolevulinate Synthetase; D008239:Lysine; D005998:Glycine