Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase
Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase
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DOI：
10.1021/bi9918745
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发表时间：
2000-02-22
期刊：
影响因子：
2.9
通讯作者：
Mildvan, AS
中科院分区：
文献类型：
--
作者：
Harris, TK;Wu, G;Mildvan, AS
MutT 酶通过在很少受攻击的 β-磷上进行亲核取代，催化三磷酸核苷 (NTP) 水解为 NMP 和 PPi。四元 E-M2+-AMPCPP-M2+ 复合物的溶液结构表明，保守残基 Glu-53、-56、-57 和 -98 位于可能充当金属配体的结合二价阳离子附近的活性位点，Lys-39 的位置促进 NMP 离去基团的离开，而 Glu-44 位于螺旋 I（残基 47-59）之前，可能稳定该结构。 螺旋向活性位点贡献四个催化残基 [Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., 和 Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]。为了测试这些提议的作用，检查了每个残基的突变对动力学参数以及 Mn2+、Mg2+ 和底物结合特性的影响。对于 E53Q 和 E53D 突变体，Mg2+ 激活酶的 k(cat) 最大降低分别为 10(4.7) 和 10(2.6) 倍，而 E44D、E56D、E56Q 和 E44Q 突变体分别观察到 97、48、25 和 14 倍降低。 Glu-98 和 Lys-39 突变对 k(cat) 的影响较小。对于野生型 MutT 及其 E53D 和 E44D 突变体，log(k(cat)) 与 pH 的关系图在上升肢上显示出 1 的极限斜率，然后出现驼峰，即在 pH 8 附近有一个明确定义的最大值，然后是一个平台，在活跃的 四元 MutT-Mg2+-dGTP-Mg2+ 复合物。 7.6 的 pK(a) 被指定为 Glu-53，在活性四元复合物中充当碱催化剂，基于 E53Q 突变体 pH 速率曲线上升肢的消失，以及其在 E53D 突变体中的恢复，(k(cat))(max) 增加 10(1.9) 倍。根据 K39Q 突变体 pi-I 速率曲线下降肢的消失，以及通过去质子化或突变去除 Lys-39 正电荷的观察结果，将 8.4 的 pK(a) 分配给 Lys-39，导致 k(cat) 降低 8.7 倍。野生型 MutT 和 E53Q 突变体的 k(cat) 值均与溶剂粘度无关，表明化学步骤可能对两者都有限速作用。 E53Q、E53D、E56Q 和 E56D 突变体在活性位点结合 Mn2+ 的能力弱 36、27、4.7 和 1.9 倍，并且表现出 2.10-、1.50-、低 1.12 倍和 1.24 倍 通过水质子的 1/T-1 值检测到，Mn2+ 的顺磁效应分别比野生型酶增强，这与金属配体的损失一致。然而，Mg2+ 和 Mn2+ 的 K-a 值表明，Glu-56 以及较小程度的 Glu-98 对活性四元复合物中的金属结合有贡献。较远但保守的残基 Glu-44 的突变对二元 E-M2+ 复合物中的金属结合或增强因子几乎没有影响。动力学损伤的 E53Q 和 E56Q 突变体的二维 H-1-N-15 HSQC 和三维 H-1-N-15 NOESY-HSQC 谱显示大部分完整的蛋白质，在突变残基附近有结构变化。在 H-1-N-15 HSQC 光谱中检测到的动力学损伤更大的 E44D 突变体的结构变化主要限于环 I-螺旋 I 基序，表明 Glu-44 稳定了活性位点区域。用 dCTP 对 E53Q、E56Q 和 E44D 突变体进行 H-1-N-15 HSQC 滴定，显示活性位点裂口内的残基化学位移发生变化，并揭示这些突变体的核苷酸结合更紧密，表明底物结合位点完整。提出了一种机制，其中 Glu-53 配位二元 MutT-M2+ 复合物中的金属，与金属解离，并对四元 MutT-M2+-dGTP-M2+ 复合物中的攻击水配体进行定向和去质子化，随后促进 PPi-M2+ 从 MutT-M2+-PPi-M2+ 产物复合物中置换。从单突变对 k(cat) 的影响来看，野生型 MutT 产生的 10(9) 倍速率加速现在可以通过酶和核苷酸结合的二价阳离子的协同效应来定量解释，其中 Glu-53 激活攻击水亲核试剂，Lys-39 促进 dGMP 离去基团的离开。
The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PPi by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M2+-AMPCPP-M2+ complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn2+, Mg2+ and substrate binding properties were examined. The largest decreases in k(cat) for the Mg2+-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed far the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg2+-dGTP-Mg2+ complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pi-I-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M2+ complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn2+ at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn2+, respectively, than the wild type enzyme as detected by 1/T-1 values of water protons, consistent with the loss of a metal ligand.However, the K-a values of Mg2+ and Mn2+ indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M2+ complexes. Two-dimensional H-1-N-15 HSQC and three-dimensional H-1-N-15 NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in H-1-N-15 HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. H-1-N-15 HSQC titrations of the E53Q, E56Q, and E44D mutants with dCTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. A mechanism is proposed in which Glu-53 coordinates the metal in the binary MutT-M2+ complex, dissociates from the metal and orients and deprotonates the attacking water ligand in the quaternary MutT-M2+-dGTP-M2+ complex, and subsequently facilitates the displacement of PPi-M2+ from the MutT-M2+-PPi-M2+ product complex. From the effects of single mutations on k(cat) the 10(9)-fold rate acceleration produced by wild type MutT can now be explained quantitatively by the cooperative effects of the enzyme- and nucleotide-bound divalent cations, with Glu-53 activating the attacking water nucleophile, and Lys-39 promoting the departure of the dGMP leaving group.