A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A.
A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A.
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在 1.8 至 1.9 A 下，从突变型单体甘氨酰胺核糖核苷酸转化酶的低 pH 和高 pH 晶体结构中观察到活性位点环的 pH 依赖性稳定。
DOI：
10.1006/jmbi.1998.1931
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Wilson,IA
中科院分区：
文献类型：
--
作者：
Su,Y;Yamashita,MM;Greasley,SE;Mullen,CA;Shim,JH;Jennings,PA;Benkovic,SJ;Wilson,IA
大肠杆菌甘氨酰胺核糖核苷酸转化酶 (GarTfase) 二聚体界面的突变破坏了观察到的野生型酶的 pH 依赖性关联，但对酶活性没有明显影响。在这里，我们评估 pH 对酶构象的影响本身是否足以解释 GarTfase 反应的 pH 依赖性。在 pH 3.5 (1.8 Å) 和 7.5 (1.9 Å) 下，Glu70Ala 突变体 GarTfase 的两个高分辨率晶体结构之间观察到 pH 依赖性构象变化。 GarTfase 中的残基 110 至 131 经历从 pH 3.5 时酶无活性的无序环转变为 pH 7.5 时酶活性的有序环-螺旋结构。这种柔性环螺旋的排序对活性位点中的催化残基、叶酸辅助因子的结合以及活性位点与溶剂的屏蔽有直接影响。有序环中 Tyr115 的主链羰基氧原子与 His108 形成氢键，从而为该关键活性位点残基提供电子和结构稳定性。动力学数据表明His108 的p Ka 实际上升高至9.2。环运动可以与 His pKa 的升高相关，但在叶酸辅因子结合后可能来自 Asp144，进一步稳定。同样在环中的 Leu118 位于对氨基苯甲酸结合位点附近，提供与辅因子 10-甲酰四氢叶酸的额外疏水相互作用。因此，酶活性的 pH 依赖性似乎是由局部活性位点重排引起的，而不是由单体-二聚体缔合引起的差异引起的。
A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme’s conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 Å) and 7.5 (1.9 Å). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the p Kaof His108 is in fact raised to 9.2. The loop movement can be correlated with elevation of the His p Ka, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.