|Title||A decade of protein engineering on ribonuclease T1--atomic dissection of the enzyme-substrate interactions.|
|Publication Type||Journal Article|
|Year of Publication||1997|
|Journal||Eur J Biochem|
|Date Published||1997 Jul 1|
|Keywords||Guanine, Hydrogen Bonding, Kinetics, Protein Engineering, Ribonuclease T1, Substrate Specificity|
During the last decade, protein engineering has been used to identify the residues that contribute to the ribonuclease-T1-catalyzed transesterification. His40, Glu58 and His92 accelerate the associative nucleophilic displacement at the phosphate atom by the entering 2'-oxygen downstream guanosines in a highly cooperative manner. Glu58, assisted by the protonated His40 imidazole, abstracts a proton from the 2'-oxygen, while His92 protonates the leaving group. Tyr38, Arg77 and Phe100 further stabilize the transition state of the reaction. A functionally independent subsite, including Asn36 and Asn98, contributes to chemical turnover by aligning the substrate relative to the catalytic side chains upon binding of the leaving group. An invariant structural motive, involving residues 42-46, renders ribonuclease T1 guanine specific through a series of intermolar hydrogen bonds. Tyr42 contributes significantly to guanine binding through a parallel face-to-face stacking interaction. Tyr45, often referred to as the lid of the guanine-binding site, does not contribute to the binding of the base.
|Alternate Journal||Eur. J. Biochem.|