Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator.
Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator.
复制标题
在变构激活剂存在和不存在的情况下，酸和 AMP 核苷酶对 AMP 进行 N-糖苷水解的过渡态结构。
DOI：
10.1021/bi00377a037
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Schramm,VL
中科院分区：
文献类型：
--
作者：
Mentch,F;Parkin,DW;Schramm,VL
宾夕法尼亚州费城坦普尔大学医学院生物化学系，1986年8月4日收到；修订稿收到1986年10月6日摘要：通过拟合实验观察到的动力学同位素效应[Parkin，D.W.，&Schramm，V.L.(19140)生物化学(本期的前一篇论文)]，研究了AMP的N-糖苷键的酸和酶水解的机理。通过改变过渡态结构并将计算的动力学同位素效应的变化与同位素效应测量的实验值进行比较，测试了过渡态计算的灵敏度。AMP酸催化水解的动力学同位素效应最好的解释是核糖环上具有相当大的氧碳阳离子特征的过渡态、与离开的腺嘌呤环上仍有显著的成键、亲水亲核剂的参与和腺嘌呤环的质子化。没有水亲核试剂预缔合的过渡态结构不能被数据消除。分析了棕色固氮菌的AMP核苷酶在没有和存在变构激活剂镁ATP的情况下对AMP的N-糖苷键的酶解作用。最好地解释动力学同位素效应的酶催化水解的过渡态包括早期SN1过渡态，其糖苷键和腺嘌呤碱基的质子化具有显著的键级。该酶强制酶结合的水分子的参与，该水分子在过渡状态下与CV有弱的结合。镁-三磷酸腺苷激活AMP核苷酶使糖苷键在过渡态的键级显著增加。核糖基中的超共轭被氧碳正离子的酶稳定所改变。这种变化与氨基酸与酶的相互作用是一致的。总而言之，这些变化稳定了一个类碳的过渡态复合体，它比没有变构激活剂时更早出现在反应途径中。除了改变过渡态结构的变构变化外，动力学同位素测量没有观察到的其他诱导效应的存在也可能增加催化速率。棕色固氮菌的单磷酸核苷酶催化AMP可逆水解为腺嘌呤和5-磷酸核糖：
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received August 4, 1986; Revised Manuscript Received October 6, 1986 abstract: The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D. W., & Schramm, V. L.(1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by varying the transition-state structure and comparing changes in the calculatedkinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Kmax approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to CV in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increasethe catalytic rate..^^. denosine monophosphatenucleosidase from Azotobacter vinelandii catalyzes the reversible hydrolysis of AMP to adenine and ribose 5-phosphate: