Conformational dynamics and solvent viscosity effects in carboxypeptidase-A-catalyzed benzoylglycylphenyllactate hydrolysis.

We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 2H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular friction-free nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site. We illustrate the approach by new spectrophotometric and pH-titration data for carboxypeptidase-A-catalyzed benzoylglycyl-L-phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 2H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180-240-nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme-peptide substrate complex with a 'tight' structure protected from the solvent. In comparison, the enzyme-ester substrate complex is 'softer', strongly coupled to the solvent, and the rate-determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.

[Effect of medium viscosity on the activity of myosin ATPase]. / Vliianie viazkosti sredy na aktivnost' miozinovoi ATFazy.

The influence of increased medium viscosity on the activity of myosin Ca2+-ATPase has been studied in 28, 45 and 60% water-sucrose solutions at 25 degrees C. In the wide range of viscosities (10 divided by 430 mp) the rate constant of ATP hydrolysis displays the negative power-law dependence on solution viscosity with an index approximately -0,5. The obtained data confirm an idea about the existence of direct connection between the low-frequency liquid relaxations and structural dynamics of proteins and enzymes.