Chemical synthesis and enzymatic properties of RNase A analogues designed to enhance second-step catalytic activity
| Autor: | Valentina Tereshko, David J. Boerema, Stephen B. H. Kent, Junliang Zhang |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
RNase P Stereochemistry Crystallography X-Ray Biochemistry Chemical synthesis RNase PH Enzyme catalysis 03 medical and health sciences Side chain Moiety Animals Amino Acid Sequence Physical and Theoretical Chemistry Binding site Phosphorylation Adenine binding Binding Sites 030102 biochemistry & molecular biology Chemistry Adenine Hydrolysis Organic Chemistry Ribonuclease Pancreatic Molecular Docking Simulation Cattle |
| Zdroj: | Organicbiomolecular chemistry. 14(37) |
| ISSN: | 1477-0539 |
| Popis: | In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 A resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology. |
| Databáze: | OpenAIRE |
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