Kaiser P M, Bonacker L, Witzel H, Holý A
Hoppe Seylers Z Physiol Chem. 1975 Feb;356(2):143-55.
Ribonuclease T2 was isolated from an Aspergillus oryzae extract. In order to define the substrate specificity, the hydrolysis of a series of 2',3'-cyclic nucleotides was measured semiquantitatively. Modifications in all positions of the bases are tolerated, as long as the base stays in the anti conformation or has a chance to return to it; bulky substituents at N-3 of the pyrimidine base lower the rate. So far the conclusion seems justified that the enzyme does not react with the substrates by specific bonds to the base, but rather by hydrophobic binding. The conformation specificity and the pH dependence of the activity support this hypothesis. The pH optima with substrates which may be positively or negatively charged are shifted to pH values at which the substrates are uncharged. This strongly indicates a hydrophobic type of interaction between base and enzyme. From the pH dependence of the kinetic parameters Km and k+2, an enzyme group with a pK of 7 (probably histidine) can be postulated. This group should interact in the protonated form with the phosphate anion. Another B.HB-system (probably two carboxylate groups) seems to be involved in the catalysis step, performing the base catalysis at the 2'-OH group and the proton catalysis at the phosphate oxygen simultaneously.
核糖核酸酶T2是从米曲霉提取物中分离得到的。为了确定底物特异性，对一系列2',3'-环核苷酸的水解进行了半定量测定。只要碱基保持反式构象或有机会恢复到反式构象，碱基所有位置的修饰都是可以接受的；嘧啶碱基N-3位的大取代基会降低反应速率。到目前为止，这样的结论似乎是合理的：该酶与底物反应不是通过与碱基形成特定的键，而是通过疏水结合。构象特异性和活性对pH的依赖性支持了这一假设。对于可能带正电或负电的底物，其最适pH值会移至底物不带电时的pH值。这有力地表明了碱基与酶之间存在疏水相互作用类型。从动力学参数Km和k+2对pH的依赖性可以推测，存在一个pK为7的酶基团（可能是组氨酸）。该基团应以质子化形式与磷酸根阴离子相互作用。另一个B.HB系统（可能是两个羧基）似乎参与了催化步骤，同时在2'-OH基团处进行碱催化，在磷酸氧处进行质子催化。
