Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering |
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Authors: | Hakoshima, Toshio Toda, Shoji Sugio, Shigetoshi Tomita, Ken-ichi Nishikawa, Satoshi Morioka, Hiroshi Fuchimura, Kayoko Kimura, Tsuyoshi Uesugi, Sei-ichi Ohtsuka, Eiko Ikehara, Morio |
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Affiliation: | Faculty of Pharmaceutical Sciences, Osaka University Osaka 565, Japan 1Faculty of Pharmaceutical Sciences, Hokkaido University Sapporo 060, Japan |
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Abstract: | Recognition by ribonuclease T1 of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr42-Asn43-Asn44-Tyr45-Gly46-Gly47-Phe48. Thesegment displays a unique folding of the polypeptide chainconsistingof a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by ahydrogen-bond network involving the side chain of Asn44, themain-chain atoms of Asn44, Gly47 and Phe48 and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn43 and Ser54. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn44 with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn44 plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn43 by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu46 to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn43 by a histidineand Asn44 by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease Ms, showed an activity and base specificitysimilar to that of the wild-type ribonuclease Ms. The segmenttherefore appears to be well conserved in several fungal ribonucleases. |
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Keywords: | specificity/ hydrogen bond/ kinetics/ reverse turn/ substructure |
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