Bastyns K, Froeyer M, Volckaert G, Engelborghs Y
Laboratory of Chemical and Biological Dynamics, University of Leuven, Belgium.
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):737-42. doi: 10.1042/bj3000737.
A computer model of the complex between G2'p5'G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2'p5'G. This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine. A barnase mutant was therefore made in which Glu-60 was replaced by Gln. This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat. For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined. The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A). This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A). For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair. Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km. In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate. The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding. The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations. Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters.
基于核糖核酸酶T1.G2'p5'G的已知结构，构建了G2'p5'G与解淀粉芽孢杆菌重组核糖核酸酶巴那斯酶复合物的计算机模型。该模型表明，保守残基Glu-60在巴那斯酶对鸟苷的特异性中起重要作用。因此，制备了一种巴那斯酶突变体，其中Glu-60被Gln取代。该突变使二核苷酸GpC和GpA的Km增加了10倍，但不改变kcat。对于ApA，kcat/Km以类似的倍数降低，但无法确定各个参数。然而，该突变对巴那斯酶作用于RNA和聚腺苷酸的kcat和Km没有影响。这表明在ApA和聚腺苷酸的情况下，底物与60位残基之间的相互作用一定不同。对于RNA，这个结论也很可能成立，但不是绝对确定，因为巴那斯酶/RNA可能是Briggs-Haldane型酶/底物对。因此，如果突变的影响仅限于底物解离速率常数（k-1）的增加，这在Km或kcat/Km中不会明显体现。鉴于聚腺苷酸的情况明确，针对该底物研究了突变型巴那斯酶的pH谱以及盐浓度对其动力学参数的影响。盐对Km的影响可以通过连锁函数概念来解释，表明聚腺苷酸结合时7-10个抗衡离子的协同解离。在高pH下，底物的结合显著降低，并且涉及的pKa在高盐浓度下大幅下降。聚腺苷酸和RNA的吸收光谱显示出pH依赖性，表明碱基堆积存在pH依赖性变化，这可能会影响催化参数。
