Kumar K, Walz F G
Department of Chemistry, Kent State University, Kent, Ohio 44242, USA.
Biochemistry. 2001 Mar 27;40(12):3748-57. doi: 10.1021/bi002837c.
Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.
进行了涉及Asn43与Asn44（第1组）或Glu46与相邻插入（第2组）的组合随机诱变，以探索核糖核酸酶T(1)（RNase T(1)）鸟嘌呤识别环的功能完善情况。在每组中筛选了400个独特的重组体，以评估它们增强RNA切割酶催化作用的能力。经过全面的筛选程序，仅发现6个变体与野生型活性相当或更高，其中包括Asn43被Gly、His、Leu或Thr取代，意外的Tyr45Ser取代以及Glu46Pro与相邻的Glu47插入。Asn43His-RNase T(1)具有与RNases Pb(1)和Fl(2)相同的环序列。活性最高的突变体均不是Asn44的单取代或Asn43和Asn44的双取代。总共纯化了13个变体，并以RNA、GpC和ApC作为底物对其进行动力学分析。GpC和RNA活性适度增强涉及k(cat)和K(M)效应。对GpC活性较低的突变体对RNA的相对活性相应更低。Asn43Gly-RNase T(1)和第2组中纯化的所有5个突变体对ApC的k(cat)/K(M)值相似，是观察到的最高值，约为野生型的10倍。特异性比[(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)]变化超过30000倍，其中Asn43His变体增加了10倍[主要是由于(k(cat)/K(M))(ApC)较低]，而Glu46Ser/(插入)Gly47变体降低了3000倍[主要是由于(k(cat)/K(M))(GpC)较低]，与野生型相比。有趣的是，Tyr45Ser变体的k(cat) (GpC)几乎比野生型大4倍，并且Pro46/(插入)Glu47 RNase T(1)的活性比具有保守Glu46的置换变体(插入)Pro47-RNase T(1)高70倍。无论如何，在800个被研究的变体中只有6个具有野生型活性这一观察结果支持了RNase T(1)鸟嘌呤识别环已实现功能完善的观点。
