Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.
Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.
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从 X 射线结构和蛋白质工程推断核糖核酸酶 T1 鸟嘌呤结合位点的构象特性。
DOI：
10.1093/protein/2.1.55
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
E. Ohtsuka
中科院分区：
文献类型：
--
作者：
T. Hakoshima;S. Toda;S. Sugio;K. Tomita;S. Nishikawa;H. Morioka;K. Fuchimura;T. Kimura;S. Uesugi;E. Ohtsuka
核糖核酸酶T1通过多齿氢键和堆积相互作用识别鸟嘌呤碱基似乎主要由一段七肽Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47-Phe48形成的短肽段介导。该片段显示出多肽链的独特折叠-由反向转角Asn44-Tyr45-Glu46-Gly47组成，由涉及Asn44的侧链、Asn44、Gly47和Phe48的主链原子和一个水分子的氢键网络稳定。该片段连接到β链的C末端，并扩展到Asn43和Ser54之间的环区域。低的值的结晶热参数的段表明，该结构具有的刚度相媲美的β-折叠片。用丙氨酸替换Asn44导致酶活性低得多，并且表明Asn44的侧链除了在维持片段结构中的作用之外，在多肽折叠中起关键作用。用丙氨酸取代Asn43以去除与鸟嘌呤碱基的弱氢键，使复合物的过渡态在37 ℃下不稳定6.3 kJ/mol。与此相反，突变的Glu46丙氨酸，以消除一个强的氢键的鸟嘌呤基地造成的不稳定的复杂的14.0千焦/摩尔。一个双突变体酶与取代的Asn43由组氨酸和Asn44由天冬氨酸，复制核糖核酸酶中发现的天然取代MS，表现出类似于野生型核糖核酸酶MS的活性和碱基特异性的片段，因此似乎是很好地保守在几种真菌核糖核酸酶。
Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.