Tan D, Ferreira G C
Department of Biochemistry and Molecular Biology, College of Medicine, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa 33612, USA.
Biochemistry. 1996 Jul 9;35(27):8934-41. doi: 10.1021/bi952918m.
5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.
5-氨基酮戊酸合酶（EC 2.3.1.37）是动物、真菌和一些细菌血红素生物合成途径中的第一种酶。它以同源二聚体形式发挥作用，需要磷酸吡哆醛作为必需辅因子。在小鼠红系5-氨基酮戊酸合酶中，赖氨酸313已被确定为与磷酸吡哆醛形成席夫碱连接的残基[费雷拉，G.C.等人（1993年）《蛋白质科学》2，1959 - 1965]，而精氨酸149是所有已知5-氨基酮戊酸合酶序列中的保守残基，对其功能至关重要[龚和费雷拉（1995年）《生物化学》34，1678 - 1685]。为了确定每个亚基是否包含独立的活性位点（即亚基内排列），或者活性位点是否位于亚基界面（即亚基间排列），体内互补研究被用于从定点的、催化无活性的小鼠5-氨基酮戊酸合酶突变体产生异二聚体。当R149A和K313A突变体在hemA - 大肠杆菌菌株中共表达时，该菌株只能在5-氨基酮戊酸存在的情况下生长，或者当它用活性5-氨基酮戊酸合酶表达质粒转化时才能生长，hemA - 大肠杆菌菌株获得了血红素原养型。纯化的K313A/R149A异二聚体混合物对底物的K(m)值与野生型酶相似，并且具有约26%的野生型酶活性，这与K313A/R149A共表达系统预期的25%的值一致。此外，对四个缺乏ALAS活性但表现出酶互补作用的酿酒酵母5-氨基酮戊酸合酶突变体进行的DNA测序表明，具有N157Y和N162S突变的突变体G101可以与具有T452R突变的突变体G220互补，具有C145R突变的突变体G205可以与具有G344C突变的突变体Ole3互补。综上所述，这些结果提供了确凿的证据，表明5-氨基酮戊酸合酶活性位点位于亚基界面，并且包含来自两个亚基的催化必需残基。
