Numata T, Suzuki A, Yao M, Tanaka I, Kimura M
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
Biochemistry. 2001 Jan 16;40(2):524-30. doi: 10.1021/bi002096f.
The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.
从苦瓜（苦瓜属）种子中分离出的核糖核酸酶MC1（RNase MC1）由190个氨基酸组成，其特征在于在尿苷的5'侧进行特异性切割。利用定点诱变来评估α4和α5螺旋之间的α4-α5环上的四个氨基酸Asn71、Val72、Leu73和Arg74对RNase MC1识别尿嘧啶碱基的贡献。制备了四个突变体，N71T、V72L、L73A和R74S，其中RNase MC1中的Asn71、Val72、Leu73和Arg74分别被烟草中一种鸟苷酸优先核糖核酸酶NW中的相应氨基酸Thr、Leu、Ala和Ser取代，并对其酶活性进行了表征。用二核苷单磷酸CpU进行动力学分析表明，突变体N-71T的Km增加了7.0倍，kcat降低了2.3倍，而突变体L73A的Km增加了14.4倍，尽管其kcat值与野生型相当。相比之下，用相应氨基酸Leu和Ser分别取代Val72和Arg74对酶活性影响很小。这一观察结果与晶体结构分析的结果一致，即Asn71和Leu73决定了RNase MC1对尿苷的特异性。通过取代氨基酸Ala、Ser、Gln和Asp进一步研究了Asn71在RNase MC1酶促反应中的作用。我们的观察结果表明，Asn71至少有两个作用：一是通过氢键进行碱基识别，二是通过与肽主链形成氢键来稳定α4-α5环的构象，这些事件可能导致含有活性位点残基的α-螺旋（α5）具有合适的取向。通过对CpG的切割评估，突变体N71T和N71S表现出从尿嘧啶特异性到鸟嘌呤特异性的显著转变，尽管它们对酵母RNA和同聚核苷酸表现出尿苷特异性。
