Karpeĭskiĭ M Ia, Moiseev G P, Bocharov A L, Bogdanova G A, Mikhaĭlov S N
Bioorg Khim. 1983 Jun;9(6):803-14.
To get insight into the origin of pyrimidine specificity of ribonuclease A, a study of the enzyme interaction with the substrate analogs having a modified nucleobase was undertaken. Pyridine and pyrimidine cyclophosphates were obtained by phosphorylation of 2'(3')-O-dibutyl stannylidene derivatives of nonprotected nucleosides in high yields. The results of kinetic and NMR studies suggested that a substrate should be locked in anti-conformation in the productive enzyme-substrate complex as it was shown for the crystalline complexes of the enzyme with pyrimidine nucleotides by X-ray analysis. The interaction between carbonyl group in position 2 of substrate nucleobase and proton accepting group of the protein (NH of Thr45) was found to be a prerequisite for the specific recognition of a substrate by the enzyme. The rate constants for transformation of lactam form (slow) and lactim form (fast) of pyrimidine substrates were estimated.
为深入了解核糖核酸酶A嘧啶特异性的起源，开展了一项关于该酶与具有修饰核碱基的底物类似物相互作用的研究。通过对未保护核苷的2'(3')-O-二丁基亚锡衍生物进行磷酸化反应，以高产率获得了吡啶和嘧啶环磷酸酯。动力学和核磁共振研究结果表明，在有活性的酶-底物复合物中，底物应呈反式构象，这正如通过X射线分析所显示的该酶与嘧啶核苷酸的晶体复合物那样。发现底物核碱基2位的羰基与蛋白质的质子接受基团（苏氨酸45的NH）之间的相互作用是该酶特异性识别底物的先决条件。估算了嘧啶底物内酰胺形式（慢）和烯醇式内酰胺形式（快）转化的速率常数。
