Santoro J, Juretschke H P, Rüterjans H
Biochim Biophys Acta. 1978 Dec 20;537(2):304-9. doi: 10.1016/0005-2795(78)90513-5.
The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.
以[¹³C₁]溴乙酸盐作为烷基化试剂制备的核糖核酸酶A衍生物Npi-[¹³C₁]羧甲基-组氨酸-119核糖核酸酶，已通过高分辨率¹³C核磁共振光谱进行了研究。在¹³C核磁共振谱中出现了两个强度相对较高的碳共振峰，它们可归属于连接到组氨酸-119和甲硫氨酸-30上的羧基，其强度比为10:1。与组氨酸-119的Npi相连的羧甲基的碳共振峰的pH依赖性在不存在和存在Cyd-2'-P时有所不同，这表明催化无活性的衍生物确实能结合核苷酸。提出了在pH 5.6时烷基化反应的机制，其中赖氨酸-41的ε-氨基作为溴乙酸羧基的结合位点，将溴亚甲基推向组氨酸-119的Npi或组氨酸-12的Ntau。
