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

Abstract

Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step.

Electron transfer with azurin at Au–SAM junctions in contact with a protic ionic melt: impact of glassy dynamics

“…15,16,20,21,[24][25][26][39][40][41][43][44][45][53][54][55]71 In the case of sluggish (viscous) environments, the slow modes of the protein's outer solution (e.g., of the water-doped [ch][dhp] melt), of the protein's peripheral (interfacial) zone, and even of the protein's metal core neighborhood may be essentially coupled, and form hierarchical (intrinsically subordinated) set of connections (see the following sections for further discussion). 15,16,20,21,[24][25][26][53][54][55][68][69][70][71][72][73] Accordingly, in the case of composite systems like ours, the classical Marcus relationship 43,45 for the outer-sphere component of the reorganization energy, l o(OS) p [A o À1 À A s À1 ] (where A o and A s are optical and static dielectric permittivity constants of the medium, respectively), is hardly applicable. 74,75 Anyway, the values of A o and A s , for choline-based PILs change little for respective binary mixtures above the [ch][dhp] mole fraction of ca.…”

Electron transfer with myoglobin in free and strongly confined regimes: disclosing diverse mechanistic role of the Fe-coordinated water by temperature- and pressure-assisted voltammetric studies

“…It is well known that additives of urea cause proteins' thermodynamic destabilization [73,79], whereas TMAO, in general, efficiently stabilizes proteins [73,79]. In numerous publications, it has been demonstrated that the trends of proteins' stabilization/destabilization generally are accompanied with the decrease/increase of a fluctuational flexibility of proteins, such that the total unfolding (lack of secondary and tertiary structures) matches the maximal flexibility [73,[80][81][82].…”

“…It is well known that additives of urea cause proteins' thermodynamic destabilization [73,79], whereas TMAO, in general, efficiently stabilizes proteins [73,79]. In numerous publications, it has been demonstrated that the trends of proteins' stabilization/destabilization generally are accompanied with the decrease/increase of a fluctuational flexibility of proteins, such that the total unfolding (lack of secondary and tertiary structures) matches the maximal flexibility [73,[80][81][82]. Furthermore, it has been well established that Mb, in general, is a rather flexible protein that is required for the realization of its function − ligand binding/release through the "gating" motion of the protein matrix throughout the region of the "ligand channel" [32][33][34][44][45][46][47][48].…”

“…If a frictional control (equations (2) and (6)) is the case, in general, it can be written [23][24][25][26][27][28][29][70][71][72][73]: 6 T.D. Dolidze et al…”

Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A

“…CPA was discovered in 1929 [18] and the first crystal of CPA was obtained from Bos taurus pancreas in 1937 [19]. It is a 35 kDa zinc-dependent metal carboxypeptidase, routinely obtained at an industrial scale from porcine or bovine pancreas [20]. It can cut off the C-terminal amino acid (especially aromatic amino acid) residues, but not proline, hydroxyproline, arginine, and lysine residues.…”