突变、动能和NMR研究机制的大肠杆菌GDP-mannose mannosyl水解酶,一种不同寻常的Nudix酶。
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马Legler点,Massiah Mildvan
突变、动能和NMR研究机制的大肠杆菌GDP-mannose mannosyl水解酶,一种不同寻常的Nudix酶。
生物化学。2002年9月3;41 (35):10834 - 48。
- PubMed ID
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12196023 (在PubMed]
- 文摘
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GDP-mannose mannosyl水解酶(GDPMH)是一个不寻常的Nudix家人,催化水解的GDP-alpha-D-mannose GDP和beta-sugar通过亲核取代碳而非磷(Legler, p . M。Massiah, m。Bessman, m . J。,Mildvan a s(2000)生物化学39岁,8603 - 8608)。使用的结构和机制的杂种狗,典型的Nudix酶作为指导,我们发现六GDPMH催化残基,三是GDPMH特有的,由动力学和结构位点突变的影响。glu - 70(对应于Glu-57杂种狗)提供了一个对这个重要的二价阳离子配体E70Q突变的影响的基础上,减少kcat 10(2.2)倍,增加了离解常数的三元E-Mn2 Mn2 + + gdp复杂的三倍,增加了K (m) Mg2 + 20倍,和减少Mn2 + 1日/ T1的顺磁影响水的质子,指示协调Mn2 +范围的变化。E70Q突变,gln - 70活性部位附近的证明是非常大型顺磁金属离子的影响在其侧链- nh2 Mn2 +组。与野生型GDPMH, pH值的影响日志(kcat / K (m) GDPmann)在37摄氏度显示一个提升肢体单位斜率,紧随其后的是一个高原的pK (a)为6.4,增加到6.7 + / - 0.1的pH值依赖的日志(kcat)。一般基催化剂被确认为一个中立的他残留的DeltaH(电离)= 7.0 + / - 0.7千卡/摩尔,pK的增加与离子强度(a),并由各四个组氨酸残基的突变GDPMH Gln。只有H124Q变异显示提升肢体的损失在pH值和日志(kcat)速度剖面,取而代之的是一个弱依赖率的氢氧根浓度,以及整体10(3.4)倍kcat下降,表明他的- 124总基地,与杂种狗不同的是,它使用Glu-53这个角色。H88Q突变体显示10(2.3)倍kcat减少,增加了4.4倍K (m) GDPmann和pH值的变化与日志(kcat)速度剖面,表明一个重要的但他的不明身份的角色- 88催化。一和二维核磁共振研究允许序列的特定作业咪唑HdeltaC, H(ε)C, N(δ)和N(ε)定义的四个组氨酸和共振的质子化作用。 The pK(a) of His-124 (6.94 +/- 0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/- 0.20). The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold. The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.