Gong J, Hunter G A, Ferreira G C
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612, USA.
Biochemistry. 1998 Mar 10;37(10):3509-17. doi: 10.1021/bi9719298.
5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.
5-氨基酮戊酸合酶（ALAS）催化非植物真核生物和一些原核生物血红素生物合成途径的第一步，即甘氨酸与琥珀酰辅酶A缩合生成辅酶A、二氧化碳和5-氨基酮戊酸。ALAS需要磷酸吡哆醛作为必需的辅因子，并以同型二聚体形式发挥作用。已发现小鼠红细胞酶中的D279在所有氨基酮戊酸合酶中保守，且似乎与天冬氨酸转氨酶中的D222同源，该残基的侧链可稳定辅因子环氮的质子化形式，从而在酶催化过程中增强辅因子的电子受体功能。ALAS中的D279A突变在标准测定条件下未检测到酶活性，保守的D279E突变使琥珀酰辅酶A的催化效率降低了30倍。D279A突变导致磷酸吡哆醛辅因子结合的解离常数增加了19倍。紫外可见光谱和圆二色光谱分析表明，D279A突变体在活性位点以不同模式结合辅因子。与野生型和D279E突变体相反，D279A突变体在结合5-氨基酮戊酸后未能催化醌型中间体的形成。重要的是，通过用辅因子类似物N-甲基-PLP重构突变体，D279A中的这一中间反应得以挽救。当用氘代甘氨酸替代甘氨酸时，野生型酶的稳态动力学同位素效应较小（kH/kD = 1.2±0.1），但D279E突变体观察到强烈的同位素效应（kH/kD = 7.7±0.3）。用ALA形成的外部醛亚胺的pH滴定表明，D279E突变使醌型形成的表观pKa从8.10增加到8.25。结果与D279通过增强辅因子的电子受体功能在氨基酮戊酸合酶催化中起关键作用的提议一致。
