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.
Goguadze
N G
NG
Institute of Inorganic Chemistry and Electrochemistry, Georgian Academy of Sciences, Tbilisi, USSR.
Hammerstad-Pedersen
J M
JM
Khoshtariya
D E
DE
Ulstrup
J
J
D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002384:Catalysis; D002942:Circular Dichroism; D006626:Hippurates / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D014783:Viscosity
D006626:Hippurates; D007773:Lactates; C066372:benzoylglycyl phenyllactate; D002268:Carboxypeptidases; D043422:Carboxypeptidases A