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

Abstract

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

Diverse role of conformational dynamics in carboxypeptidase A‐driven peptide and ester hydrolyses: Disclosing the “Perfect Induced Fit” and “Protein Local Unfolding” pathways by altering protein stability

“…Here, we note that under standard conditions (no urea or TMAO added), the results for k cat and K m regarding both substrates are within the range of published results, 3,4,12,[17][18][19]45 with a much larger spread for the latter parameter which, seemingly, is caused by the following : (i) determination of K m through the Lineweaver-Burk procedure, in general, is connected to the extrapolation of experimental points over larger numerical windows and, (ii) in this particular case, the large uncertainty in choosing the ''valid'' intervals of substrate concentration when the method of ''initial slopes'' is applied, cf. Figures S3 and S4 ; note large positive value for the latter one) for the hydrolyses of Bz-Gly-OPhe, an even more notable deviation from the published results (ranging within 40 to 54 kJ mol 21 for DH a(eff) , and occurring around 240 J K 21 mol…”

“…Kinetic traces were recorded at a fixed wavelength of 260 nm, where the spectral changes due to both the hydrolytic processes are optimal. 19,45 The working concentration for the substrates (1 mM) was such that the absorbance in the stopped-flow optical cell did not exceed 1. The necessary calibrating (static) spectra of substrates and the hydrolysis products were recorded on a Cary 3 (Varian) UV-Vis instrument.…”

“…[3][4][5][6][12][13][14][15][16][20][21][22][23][24][25][26][27] The ''promoted water'' pathway entails direct participation of Zn 21 -activated water in a rate-determining step as a nucleophile with the carboxylate group of Glu270 acting as a general base (Scheme 1), whereas the ''anhydride intermediate'' pathway implies direct nucleophilic attack by Glu270 (Scheme 2), with water to be involved in the following stage (for the detailed discussion, see Results and Discussion). Notwithstanding that the mechanistic difference for peptide and ester substrates has been discussed numerous times, [3][4][5][6]12,13,18,19,26,27 definitely this problem requires further clarification, implying both the adequate classification and theory-based description, on the grounds of matching current concepts (vide infra). Much inconvenience on the way of experiment-aided mechanistic analysis comes from the fact that small specific probing substrates, both peptide and ester, for which combined kinetic studies usually give decisive mechanistic information (see, e.g., Refs.…”

“…3,6,8,[23][24][25][26][27][28] This situation is rather distinct from some other classes of model enzymes such as, e.g., serine hydrolases for which the major mechanistic pattern has been firmly established. 29,30 Among other complexities, extremely diverse kinetic manifestations showing up through the pH profiles, [3][4][5][6]12 inhibition/activation patterns (due to substrates or other effectors), [3][4][5][6]12,13,22 cryospectroscopic (intermediate trapping) experiments, 6,14,16 and viscosity impact observations, [17][18][19] should be mentioned. Interestingly, rather diverse performance has been observed not only between two families of more natural yet synthetic (''probing'') peptide and the counterpart ester (depsi-peptide) substrates but also within the substrates of either of these types that may differ in the chain length and/or residue specificity.…”

Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions

“…Assuming that ΔH Ã a ¼ 7.2 kJ mol −1 (because still V AB ≪ λ o ∕4; see above), one obtains H aðηÞ ¼ 15.4 − 7.2 ¼ 8.2 ðkJ mol −1 Þ, a value that can be attributed to the accompanying relaxation process (es) of the slowly fluctuating protein/water/SAM environment (see also refs. 24,28,54). Interestingly, the value of ΔH aðEXPÞ for n ¼ 10 (the transition point between two regimes; Fig.…”