Substrate specificity of solvent viscosity effects in carboxypeptidase A catalyzed peptide hydrolysis

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…”

“…[3][4][5][6][12][13][14][15][16][17][18][19][20][21][22] Perhaps, the most convincing experimental finding of this kind was the fact that the hydrolytic process for Bz-Gly-OPhe displays an essential dependence on the solution viscosity (varied through the sucrose and glycerol additive) whereas for BzGly-Phe it does not. [17][18][19] Clearly, this outstanding phenomenon cannot be masked by the side effects of the substrate activation or inhibition. In the earlier work, 3,6,[20][21][22] it has been conjectured that an ''extra'' proton at the nitrogen of the substrate scissile bond, in addition, interacts with a carboxyl group of Glu270 in the substrate binding step (in contrast to Bz-Gly-OPhe, for which this interaction is excluded because of the substitution of [-NH-] by [-O-]).…”

“…As already mentioned in the Introduction, Bz-Gly-Phe and Bz-Gly-OPhe are prototype model substrates for CPA and have long been used for mechanistic studies using both, experimental 4,12,[17][18][19]22,45 and computational [23][24][25][26][27] techniques. Although a current outline of the previous experimental work implies two distinct mechanisms for typical (and specific) peptide and ester substrates such are Bz-Gly-Phe and Bz-Gly-OPhe (vide supra), recent, most sophisticated computational studies 26,27 deduced that not only the same overall mechanism (the ''promoted water'' pathway) is operative in both cases but also the rate-determining step, that is the nucleophilic attack of a scissile bond by water, accompanied by the concerted proton transfer to Glu270 (general base catalysis), is the same.…”

“…[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.…”