Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates.
5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.
Hunter
G A
GA
Department of Biochemistry and Molecular Biology, College of Medicine, Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33612, USA.
Ferreira
G C
GC
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000378:metabolism; D002384:Catalysis; D005998:Glycine / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D016296:Mutagenesis; D013057:Spectrum Analysis; D014316:Tritium
D000214:Acyl Coenzyme A; D014316:Tritium; D000622:Aminolevulinic Acid; C012046:succinyl-coenzyme A; D000624:5-Aminolevulinate Synthetase; D008239:Lysine; D005998:Glycine