Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an am'/> PROTON NMR STUDIES OF CYTOCHROME C PEROXIDASE MUTANT N82A - HYPERFINE RESONANCE ASSIGNMENTS, IDENTIFICATION OF TWO INTERCONVERTING ENZYME SPECIES, QUANTITATING THE RATE OF INTERCONVERSION, AND DETERMINATION OF EQUILIBRIUM CONSTANTS
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首页> 外文期刊>Biochemistry >PROTON NMR STUDIES OF CYTOCHROME C PEROXIDASE MUTANT N82A - HYPERFINE RESONANCE ASSIGNMENTS, IDENTIFICATION OF TWO INTERCONVERTING ENZYME SPECIES, QUANTITATING THE RATE OF INTERCONVERSION, AND DETERMINATION OF EQUILIBRIUM CONSTANTS
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PROTON NMR STUDIES OF CYTOCHROME C PEROXIDASE MUTANT N82A - HYPERFINE RESONANCE ASSIGNMENTS, IDENTIFICATION OF TWO INTERCONVERTING ENZYME SPECIES, QUANTITATING THE RATE OF INTERCONVERSION, AND DETERMINATION OF EQUILIBRIUM CONSTANTS

机译：细胞色素C过氧化物酶突变体N82A的质子NMR研究-超精细共振分配，两种互转换酶种类的鉴定，互转化率的确定以及平衡常数的测定

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The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82 --> Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme, For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena, First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J.D., et al. (1994) Eur. J. Biochem. 224, 81-87], the Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN, Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/-53) s(-1). The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: Delta H = 60 kJ mol(-1); Delta S = 0.20 kJ K-1 mol(-1).
机译：已经通过质子NMR光谱研究了带有突变Asn82→Ala82（[N82A] CcPCN）的面包酵母酵母细胞色素C过氧化物酶突变体的氰化物连接形式。这种突变改变了一个氨基酸，该氨基酸与His52形成氢键，His52是远端的组氨酸残基，在血红素口袋中与血红素结合的配体相互作用。 His52是对细胞色素C过氧化物酶正常功能至关重要的残基。通过与野生型天然酶的1-D和NOESY光谱比较，对突变体的氰化物连接形式进行了质子超精细共振分配。对于[N82A] CcPCN，质子NMR光谱揭示了两个重要现象，第一，相似公布有关相关突变体[N82D] CcPCN的结果[Satterlee，JD等。 （1994）Eur。 J.生物化学。 [224，81-87]，Ala82突变破坏了His52与血红素连接的CN之间的氢键，其次，在解析的酶的质子谱中，解析出的24个超精细位移共振中有四个共振倍增，导致了血红素的概念活动站点环境在非常局部的范围内是动态微异构的。在纯酶制剂中检测到两种磁性不等价的酶形式。温度变化会导致两种酶形式相互转换。磁化转移实验进一步证明了酶形式之间的这种相互转化，并已用于确定相互转化的速率为250（+/- 53）s（-1）。 20℃下的平衡常数为1.5。在5到29摄氏度之间的各种温度下计算出平衡常数，得出以下值：Delta H = 60 kJ mol（-1）; δS = 0.20 kJ K-1 mol（-1）。

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