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Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations.

ABSTRACT: Recent QM/MM analyses of proton transfer function of human carbonic anhydrase II (CAII) are briefly reviewed. The topics include a preliminary analysis of nuclear quadrupole coupling constant calculations for the zinc ion and more detailed analyses of microscopic pK(a) of the zinc-bound water and free energy profile for the proton transfer. From a methodological perspective, our results emphasize that performing sufficient sampling is essential to the calculation of all these quantities, which reflects the well solvated nature of CAII active site. From a mechanistic perspective, our analyses highlight the importance of electrostatics in shaping the energetics and kinetics of proton transfer in CAII for its function. We argue that once the pK(a) for the zinc-bound water is modulated to be in the proper range (approximately 7.0), proton transfer through a relatively well solvated cavity towards/from the protein surface (His64) does not require any major acceleration. Therefore, although structural details like the length of the water wire between the donor and acceptor groups still may make a non-negligible contribution, our computational results and the framework of analysis suggest that the significance of such "fine-tuning" is likely secondary to the modulation of pK(a) of the zinc-bound water. We encourage further experimental analysis with mutation of (charged) residues not in the immediate neighborhood of the zinc ion to quantitatively test this electrostatics based framework; in particular, Phi analysis based on these mutations may shed further light into the relative importance of the classical Grotthus mechanism and the "proton hole" pathway that we have proposed recently for CAII.

SUBMITTER: Riccardi D

PROVIDER: S-EPMC6787916 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations.

Riccardi Demian D Yang Shuo S Cui Qiang Q

Biochimica et biophysica acta 20090811 2

Recent QM/MM analyses of proton transfer function of human carbonic anhydrase II (CAII) are briefly reviewed. The topics include a preliminary analysis of nuclear quadrupole coupling constant calculations for the zinc ion and more detailed analyses of microscopic pK(a) of the zinc-bound water and free energy profile for the proton transfer. From a methodological perspective, our results emphasize that performing sufficient sampling is essential to the calculation of all these quantities, which r ...[more]

PMID: 19679196

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Project description:Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

Project description:A combined quantum mechanical/molecular mechanical (QM/MM) potential function is used in a thermodynamic integration approach to calculate the pK(a) of residue 66 in two mutants (V66E, V66D) of Staphylococal nuclease relative to solution. Despite the similarity in chemical nature and experimentally measured pK(a) of the two buried titritable residues, the behaviors of the two mutants and the computed pK(a) values vary greatly in the simulations. For Glu66, the side chain is consistently observed to spontaneously flip out from the protein interior during titration, and the overall protein structure remains stable throughout the simulations. The computed pK(a) shifts using conventional sampling techniques with multiple nanoseconds per lambda window (Set A and B) are generally close to the experimental value, therefore indicating that large-scale conformational rearrangements are not as important for V66E as suggested by the recent study of Warshel and co-worker. For Asp66, by contrast, flipping of the shorter side chain is not sufficient for getting adequate solvent stabilization of the ionized state. As a result, more complex behaviors such as partial unfolding of a nearby beta-sheet region is observed, and the computed pK(a) shift is substantially higher than the experimental value unless Asp66 is biased to adopt the similar configurations as Glu66 in the V66E simulations. Collectively, these studies suggest that the lack of electronic polarization is not expected to be the dominant source of error in microscopic pK(a) shift calculations, while the need of enhanced sampling is more compelling for predicting the pK(a) of buried residues. Furthermore, the comparison between V66E and V66D also highlights that the microscopic interpretation of similar apparent pK(a) values and effective "dielectric constants" of proteins can vary greatly in terms of the residues that make key contributions and the scale of structural/hydration response to titration, the latter of which is difficult to predict a priori. Perturbative analyses of interactions that contribute to the titration free energy point to mutants that can be used to verify the microscopic mechanisms of titration in V66E/D SNase proteins.

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Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations.

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Proton transfer function of carbonic anhydrase: Insights from QM/MM simulations.

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