Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Biochemistry. 2013 Apr 23;52(16):2783-92. doi: 10.1021/bi4001276. Epub 2013 Apr 11.
Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.
先前我们曾表明，酵母香叶基香叶基二磷酸合酶（GGPPS）在涉及二聚化的第一个 N 端螺旋缺失时变为无活性单体。这就提出了一些问题，即为什么二聚化对于 GGPPS 活性是必需的，以及二聚体界面中的哪些氨基酸对于二聚化介导的活性是必需的。根据 GGPPS 晶体结构，位于一个亚基的螺旋 F 中的三个氨基酸（N101、N104 和 Y105）位于另一个亚基的活性部位附近。如这里所示，当这些残基分别被 Ala 取代时，没有引起显著的活性变化，但 N101A/Y105A 和 N101A/N104A 而不是 N104A/Y105A 显示出明显降低的 k(cat) 值（200-250 倍）。三重突变体 N101A/N104A/Y105A 没有检测到活性，尽管这些突变体中保留了二聚体。由于 N101 和 Y105 分别与另一个亚基中的 H139 和 R140 形成氢键，因此我们生成了 H139A/R140A 双突变体，并发现其失活并变成单体。因此，由于缺少氢键网络，这些多重突变显然会影响催化部位的完整性。此外，高度保守的螺旋 F 上的 Met111 对于二聚体形成和酶活性也是必需的。当 Met111 被 Glu 取代时，带负电荷的排斥作用将二聚体的一半转化为单体。总之，通过 N101 维持底物结合稳定性的氢键和二聚体界面中 M111 的疏水相互作用对于酵母 GGPPS 的活性是必需的。
