A Nearly Isosteric Photosensitive Amide-Backbone Substitution Allows Enzyme Activity Switching in Ribonuclease S

Citations Over TimeTop 12% of 2007 papers

Abstract

psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.

Related Papers

• → The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate(1998)37 cited
• → Nuclear magnetic resonance studies of the structure and binding sites of enzymes(1969)169 cited
• → A kinetic analysis of the pH-dependence of the action of bovine pancreatic ribonuclease A on cytidine 2′:3′-phosphate(1960)19 cited
• → Activity of Bovine Pancreatic Ribonuclease A in Mixed Solvent Systems(1960)18 cited
• → Does 3′‐cytidine monophosphate bound to ribonuclease a acquire syn‐conformation?(1977)7 cited