Gutte B
J Biol Chem. 1975 Feb 10;250(3):889-904.
A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.
通过固相法合成了核糖核酸酶S蛋白的一种70个残基的类似物。它是通过去除第21至25位的氨基末端以及36至40、58至73、87至96和113至114片段而获得的。插入了四个残基以连接由缺失形成的末端。未参与缺失的半胱氨酸残基被丙氨酸或亮氨酸残基取代。合成的多肽通过凝胶过滤分离为二聚体和单体。两个组分都通过离子交换色谱进一步纯化。使用RNA作为底物时，70个残基的二聚体S蛋白类似物的比活性约为4%。它还能切割核糖核酸酶A的其他底物，如5'-(3'-胞苷酰基)-鸟苷、5'-(3'-尿苷酰基)-鸟苷和聚胞苷酸。70个残基类似物的单体活性较低，但显示出与二聚体相同的底物特异性。发现合成的S蛋白类似物的两个组分仅催化核糖核酸酶A机制的转磷酸化步骤，并且在水解步骤中即使有活性也非常低。添加天然S肽或S蛋白不会增加转磷酸化反应的活性，但会大大提高水解步骤的反应速率。在S肽存在下，使用环胞苷2':3'-单磷酸作为底物时，70个残基的单体和二聚体S蛋白、70个残基的单体和二聚体S蛋白类似物的活性均约为8%。合成的S蛋白类似物与天然S蛋白的单体和二聚体混合物对该底物产生了更高的活性（分别为151%和74%），尽管两种复合物的两个组分中天然酶的氨基末端部分（包括His 12）都缺失了。70个残基的S蛋白类似物对DNA完全无活性，并且（有一个例外）对核糖核酸酶T1的底物也无活性。合成类似物在转磷酸化步骤中的底物特异性与天然核糖核酸酶A的底物特异性密切一致，这表明尽管母体分子的一级结构发生了剧烈变化，但活性位点的构型仍有显著的保守性。讨论了这些结果对核糖核酸酶A作用机制的可能影响。
