Total chemical synthesis of ribonuclease A and designed analogues with enhanced catalytic activity

Reproducible total chemical synthesis of enzyme molecules provides the chemist with complete control over the structural make-up of the enzyme down to the level of individual atoms. Total chemical synthesis is used here to study the structure-function relationship of bovine pancreatic ribonuclease A in ways inaccessible to traditional biochemistry. Using a combination of solid phase peptide synthesis and native chemical ligation, the practical and reproducible total chemical synthesis of the [D83A]RNase A enzyme molecule is presented. Full characterization of the resulting molecule showed that the molecule was highly pure and exhibited the correct three-dimensional fold and disulfide pairings, and the full expected activity.; This synthetic scheme was utilized to prepare three non-natural analogues of the enzyme molecule containing covalently attached adenine moieties. These analogues were designed to test the hypothesis that the dynamics of the histidine 119 side chain detrimentally affects the catalysis of the second, hydrolysis step. In each of the synthetic enzyme analogues, adenine moieties were covalently attached to the enzyme near the adenine binding site. By covalently filling the adenine binding site, the dynamics of the histidine 119 side chain were expected to be altered in such a way that the enzyme is essentially partially promoted along the reaction coordinate by pre-forming the active conformation needed to catalyze the hydrolysis reaction.; As designed, two of the three enzyme analogues exhibited an increased rate of hydrolysis. Decreases in the rate of the first, transphosphorylation step were also observed, as predicted. These results were interpreted in light of the proposed hypothesis and information contained in the literature regarding the mechanism of RNase A, and provide strong evidence that the dynamics of the histidine 119 side chain play an important role in the hydrolysis step of catalysis. This work successfully demonstrates the practical reproducible synthesis of the RNase A enzyme molecule, as well as the utility of total chemical synthesis to the study of enzyme structure-function relationships.