We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 'H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular frictionfree 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-t-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 'H 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.Protein dynamics is spanned by broad time ranges extending from sub-picoseconds to milliseconds, and even seconds [l-41. The fastest limits are represented by lattice-like atomic motion [ l -31, the slowest by denaturation or subunit association, where large protein segments are rearranged. . This points towards solvent-mediated strong damping of the conformational relaxation. These modes must therefore be associated with the vibrationally dispersive solvent relaxation properties. Among these, viscosity has come to stand out prominently.Low-frequency dielectric 'loss' is a universal feature of correlated nuclear motion in liquids and disordered solids [29], associated with diverse properties such as dielectric relaxation, viscoelasticity, etc., and including conformational protein motion. Relaxation times are determined by the thermalization rates along the assembly of dissipative coordinates, subsequent to external perturbations. The dissipative, o r frictional features are associated with interaction or collision with the nuclear fragments, and collectively grouped into what is denoted as a microviscosity function [30]. This function can be dominated by external solvent collisional properties for processes involving protein surface fragments since liquid 'hole' formation in the solvent near the protein surface must precede the surface group motion. The microviscosity, however, differs from the bulk 'kinematic' viscosity, even though both quantities represent intermolecular collisions, liquid hole formation, etc. [30]. The external solvent dependence is thus

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