Urea's Effect on the Ribonuclease A Catalytic Efficiency: A Kinetic, ^sup 1^H NMR and Molecular Orbital Study

• Published in: The Protein Journal 2013-02, Vol.32 (2), p.118
• Main Authors: Almarza, Jorge, Rincón, Luis, Bahsas, Alí, Pinto, María Angela, Brito, Francisco
• Format: Article
• Language: English
• Subjects: Amino acids; Biochemistry; Catalysis; Enzymes; Inactivation; Kinetics; Molecular biology; Protein folding; Proteins; Ribonucleic acid; RNA; Theoretical analysis; Urea
• ISSN: 1572-3887; 1573-4943
• DOI: 10.1007/s10930-013-9468-3

Understanding of protein-urea interactions is one of the greatest challenges to modern structural protein chemistry. Based in enzyme kinetics experiments and ^sup 1^H NMR spectroscopic analysis we proposed that urea, at low concentrations, directly interacts with the protonated histidines of the active center of RNase A, following a simple model of competitive inhibition. These results were supported by theoretical analysis based on the frontier molecular orbital theory and suggest that urea might establish a favorable interaction with the cationic amino acids. Our experimental evidence and theoretical analysis indicate that the initials steps of the molecular mechanism of Urea-RNase A interaction passes through the establishment of a three center four electron adduct. Also, our results would explain the observed disruption of the ^sup 1^H NMR signals corresponding to H12 and H119 (involved in catalysis) of the RNase A studied in the presence of urea. Our interaction model of urea-amino acids (cationic) can be extended to explain the inactivation of other enzymes with cationic amino acids at the active site.[PUBLICATION ABSTRACT]