Nachman J, Miller M, Gilliland G L, Carty R, Pincus M, Wlodawer A
BRI Basic Research Program, NCI-Frederick Cancer Research Facility, Maryland 21701.
Biochemistry. 1990 Jan 30;29(4):928-37. doi: 10.1021/bi00456a012.
Crystal structures of two forms of ribonuclease A with deoxynucleosides covalently bound to respectively His 12 and His 119 have been solved. One form, T-H12-RNase, has a deoxythymidine bound to N epsilon 2 of His 12, while the other one, U-H119-RNase, has a deoxyuridine bound to N delta 1 of His 119. The two crystal forms are nearly isomorphous, with two molecules in the asymmetric unit. However, the modified ribonucleases differ both in their enzymatic activities and in the conformation of the catalytic site and of the deoxynucleoside-histidine moiety. T-H12-RNase is characterized by complete loss of enzymatic activity; in this form the deoxynucleoside completely blocks the catalytic site and forms intramolecular contacts with residues associated with both the B1 and B2 sites. U-H119-RNase retains 1% of the activity of the unmodified enzyme, and in this form His 119 adopts a different orientation, corresponding to the alternate conformation reported for this residue; the deoxynucleoside-histidine moiety points out of the active site and does not form any contacts with the rest of the protein, thus allowing partial access to the catalytic site. On the basis of these structures, we propose possible mechanisms for the reactions of bromoacetamido nucleosides with ribonuclease A.
已解析出两种形式的核糖核酸酶A与分别共价结合到组氨酸12和组氨酸119上的脱氧核苷的晶体结构。一种形式，T-H12-核糖核酸酶，有一个脱氧胸苷结合到组氨酸12的Nε2上，而另一种形式，U-H119-核糖核酸酶，有一个脱氧尿苷结合到组氨酸119的Nδ1上。这两种晶体形式几乎是同晶型的，不对称单元中有两个分子。然而，修饰后的核糖核酸酶在酶活性以及催化位点和脱氧核苷-组氨酸部分的构象上都有所不同。T-H12-核糖核酸酶的特征是酶活性完全丧失；在这种形式下，脱氧核苷完全阻断了催化位点，并与与B1和B2位点相关的残基形成分子内接触。U-H119-核糖核酸酶保留了未修饰酶1%的活性，在这种形式下，组氨酸119采取了不同的取向，对应于该残基报道的交替构象；脱氧核苷-组氨酸部分指向活性位点之外，不与蛋白质的其余部分形成任何接触，从而允许部分进入催化位点。基于这些结构，我们提出了溴乙酰氨基核苷与核糖核酸酶A反应的可能机制。
