The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding
Related Experiment Video
Updated: Jul 19, 2026

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 17, 2010
1Department of Laboratory Medicine and Pathology, and Graduate Program in Biophysical Sciences, University of Minnesota, Minneapolis, MN 55455, USA.
Abstract:
We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.
Related Concept Videos
Introduction to Mechanisms of Enzyme Catalysis
Spin–Spin Coupling: Three-Bond Coupling (Vicinal Coupling)
The extent of coupling depends on the C‑C bond length, the two H‑C‑C angles, any electron-withdrawing substituents, and the dihedral angle between the involved orbitals. The...
Enzymes
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
Induced-fit Model
Enzymes exhibit substrate specificity, meaning that they can only bind to certain substrates. This is mainly determined by the shape and chemical characteristics of...
Catalytically Perfect Enzymes

