Evidence for a two-base mechanism involving tyrosine-265 from arginine-219 mutants of alanine racemase.
Evidence for a two-base mechanism involving tyrosine-265 from arginine-219 mutants of alanine racemase.
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涉及来自丙氨酸消旋酶精氨酸 219 突变体的酪氨酸 265 的二碱基机制的证据。
DOI：
10.1021/bi982924t
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Toney,MD
中科院分区：
文献类型：
--
作者：
Sun,S;Toney,MD
研究发现，在嗜热脂肪芽孢杆菌的丙氨酸消旋酶结构中，一个带正电的残基R219与磷酸吡哆醛的吡啶氮相互作用[Shaw et al. (1997)Biochemistry 36, 1329−1342]。研究人员对R219K、R219A和R219E三个位点突变体进行了表征，并将其与野生型酶（野生型酶）进行了比较，以研究R219在催化中的作用。R219K突变在功能上是保守的，保留了约25%的WT活性。R219A和R219E突变分别使酶活性降低约100倍和1000倍。这些结果表明，在这个位置上带正电的残基是有效催化所必需的。R219和Y265通过氢键H166连接。R219突变体表现出相似的动力学同位素效应趋势：主同位素效应增加（1.5 ~ 2倍），溶剂同位素效应不变；溶剂同位素效应增加（1.5 ~ 2倍），主同位素效应不变。这些结果支持涉及Y265和K39的双碱基外消旋机制。他们还认为Y265通过H166氢键网络选择性地受到R219突变的干扰。pH值从7.1 ~ 7.4 （WT和R219K）上升到9.5 ~ 10.4 (R219A和R219E)，从7.3上升到9.9 ~ 10.4。负责这种电离的基团可能是Y265的酚羟基，其pkas在WT中受到h166介导的与R219的相互作用的静电干扰。在510 nm处积累了一个吸光度带，表明醌类中间体仅与R219E在2→1方向上，这为涉及Y265的双碱机制提供了额外的证据。
A positively charged residue, R219, was found to interact with the pyridine nitrogen of pyridoxal phosphate in the structure of alanine racemase fromBacillus stearothermophilus[Shaw et al. (1997)Biochemistry 36, 1329−1342]. Three site-directed mutants, R219K, R219A, and R219E, have been characterized and compared to the wild type enzyme (WT) to investigate the role of R219 in catalysis. The R219K mutation is functionally conservative, retaining ∼25% of the WT activity. The R219A and R219E mutations decrease enzyme activity by approximately 100- and 1000-fold, respectively. These results demonstrate that a positively charged residue at this position is required for efficient catalysis. R219 and Y265 are connected through H166 via hydrogen bonds. The R219 mutants exhibit similar kinetic isotope effect trends: increased primary isotope effects (1.5−2-fold) but unchanged solvent isotope effects in thel→ddirection and increased solvent isotope effects (1.5−2-fold) but unchanged primary isotope effects in thed→ldirection. These results support a two-base racemization mechanism involving Y265 and K39. They additionally suggest that Y265 is selectively perturbed by R219 mutations through the H166 hydrogen-bond network. pH profiles show a large pKashift from 7.1−7.4 (WT and R219K) to 9.5−10.4 (R219A and R219E) forkcat/KM, and from 7.3 to 9.9−10.4 forkcat. The group responsible for this ionization is likely to be the phenolic hydroxyl of Y265, whose pKais electrostatically perturbed in the WT by the H166-mediated interaction with R219. Accumulation of an absorbance band at 510 nm, indicative of a quinonoid intermediate, only in thed→ldirection with R219E provides additional evidence for a two-base mechanism involving Y265.