Hunter G A, Ferreira G C
Department of Biochemistry and Molecular Biology, College of Medicine, Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33612, USA.
Biochemistry. 1999 Mar 23;38(12):3711-8. doi: 10.1021/bi982390w.
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.
5-氨基乙酰丙酸合酶催化甘氨酸和琥珀酰辅酶A缩合形成辅酶A、二氧化碳和5-氨基乙酰丙酸。这是动物和某些细菌中血红素生物合成的第一个关键步骤。成熟小鼠红系5-氨基乙酰丙酸合酶的赖氨酸313（K313）与磷酸吡哆醛辅因子形成席夫碱连接。在甘氨酸和琥珀酰辅酶A存在的情况下，在纯化的小鼠红系δ-氨基乙酰丙酸合酶的可见光谱中瞬时观察到醌型中间体吸收。用甘氨酸、组氨酸或精氨酸取代K313的突变酶在甘氨酸和琥珀酰辅酶A存在下没有醌型中间体形成的光谱证据。野生型5-氨基乙酰丙酸合酶在产物5-氨基乙酰丙酸存在下还形成稳定的醌型中间体。只有K313保守突变为组氨酸或精氨酸会产生与5-氨基乙酰丙酸形成醌型中间体的变体。然而，这些突变体的醌型中间体吸收明显低于野生型酶。虽然野生型酶催化[2-³H₂]-甘氨酸中的氚损失，但K313突变为甘氨酸会导致这种活性丧失。用5-氨基乙酰丙酸与野生型酶结合形成的醌型中间体进行滴定表明，醌型中间体通过转移单个质子形成，其pK为8.1±0.1。K313保守突变为组氨酸会将该值提高到8.6±0.1。我们提出，K313作为一般碱催化剂，在5-氨基乙酰丙酸合酶催化循环中促进醌型中间体的形成。
