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首页> 外文期刊>Biochemistry >Enzyme-substrate interactions with an antibiotic resistance enzyme: Aminoglycoside nucleotidyltransferase(2 '')-Ia characterized by kinetic and thermodynamic methods
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Enzyme-substrate interactions with an antibiotic resistance enzyme: Aminoglycoside nucleotidyltransferase(2 '')-Ia characterized by kinetic and thermodynamic methods

机译：酶 - 底物与抗生素抗性酶的相互作用：氨基糖苷核苷三烷基转移酶（2''） - 以动力学和热力学方法为特征

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摘要

Aminoglycoside nucleotidyltransferase(2")-Ia is one of the most often detected enzymes in aminoglycoside-resistant bacteria. Despite its prevalence, little biochemical and biophysical work has been reported for this enzyme. In the current study, substrate specificity and temperature dependence of k(cat) are determined by kinetic assays. Dissociation constants and thermodynamic properties of enzyme-substrate complexes are determined by isothermal titration calorimetry, electron paramagnetic resonance, and fluorescence spectroscopy. Kinetic studies show that aminoglycosides with 2'-NH2 are better substrates (higher k(cat)/K-m) than ones with 2'-OH when magnesium(II) is used as the catalytically required divalent cation. The activity is reduced 10-fold for substrates with 2'-NH2 when manganese(II) replaces magnesium as the required metal. However, kanamycin A, which has a 2'-OH, shows a much smaller decrease in activity when manganese substitutes for magnesium as the divalent cation. Temperature dependence studies show the activation energy of catalysis to be 19.2 kcal/mol and the temperature optimum between 30 and 32 degrees C. The binding of the aminoglycoside substrate tobramycin to the enzyme occurs with a favorable enthalpy which compensates for a large entropic penalty to yield a negative Delta G value for the complex formation. Enthalpy of binding is less exothermic in the presence of metal-nucleotide. However, due to the more favorable entropy, a more favorable Delta G is observed for the formation of the enzyme-metalnucleotide:aminoglycoside complex. Tobramycin binds to ANT(2") with a dissociation constant of 0.6 mu M, which is further reduced by 3-fold when metal-nucleotide is present. Binding of ATP to the enzyme is determined to be very weak in the absence of a divalent cation, and becomes 2 orders of magnitude tighter when magnesium or manganese is present. Binding studies also show that, in addition to binding to the enzyme in the form of metal-nucleotide complex, a second catalytically required metal binds to an additional site on the enzyme.
机译：氨基糖苷核苷转移酶（2“） - IA是抗性甘氨酸细菌中最常检测到的酶之一。尽管它普遍存在，但已经向该酶报告了很少的生化和生物物理作品。在目前的研究中，碱性特异性和k的温度依赖性。 （猫）由动力学测定决定。酶 - 衬底复合物的解离常数和热力学性质通过等温滴定热量，电子顺磁共振和荧光光谱法测定。动力学研究表明，具有2'-NH2的氨基糖苷是更好的基材（较高的K. （猫）/ km）当镁（II）用作催化所需的二价阳离子时比2'-OH。当锰（II）替代镁时，活性减少10倍的底物。所需的金属。然而，当锰替代镁作为二价阳离子时，Kanamycin A显示出少数少数较小的活性降低。TEMP蚀刻依赖性研究显示催化的活化能量为19.2kcal / mol，温度最佳30至32℃。氨基甘氨酸底物蛋白与酶的结合发生在良好的焓，这补偿了额外的熵罚球复杂地层的负ΔG值。在金属核苷酸存在下，结合的焓较低。然而，由于熵更有利，观察到更有利的δg用于形成酶 - 核苷酸：氨基糖苷复合物。用0.6μm的解离常数与蚂蚁（2“）结合，当存在金属核苷酸时进一步减少3倍。在没有二价的情况下，确定ATP与酶的结合是非常弱的当存在镁或锰时，阳离子变为2个级别。结合研究还表明，除了以金属核苷酸复合物的形式结合酶之外，第二催化所需的金属与另外的部位结合酶。

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