Computer simulation of the carbon dioxide/bicarbonate interconversion step in human carbonic anhydrase I

Aaqvist, Johan; Fothergill, Michael; Warshel, Arieh

doi:10.1021/ja00055a036

Cited by 59 publications

(44 citation statements)

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“…Note that small dielectric reorganization in proteins was invoked to explain a variety of kinetic data for charge transfer reactions (1,3,4,(46)(47)(48)(49)(50)(51) and was inferred from molecular simulations (52)(53)(54)(55)(56)(57)(58)(59)(60). Our direct measurements now provide evidence supporting this hypothesis.…”

Section: Discussionsupporting

confidence: 69%

Low dielectric response in enzyme active site

Mertz

Krishtalik

2000

Proc. Natl. Acad. Sci. U.S.A.

125

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The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of ␣-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. Many enzymatic reactions are charge transfer processes. Their activation free energy is affected by the dielectric properties of the milieu of the reacting groups (1). It has been suggested that small dielectric response at the active site could be a physical reason for high catalytic activity (2-4).Direct measurements of local dielectric properties at enzyme active sites have never been made. Most of the current knowledge is based on circumstantial evidence. Different existing data can be used to support opposing points of view. For instance, the average static dielectric constant of proteins s Ϸ 4 was estimated from measurements on dry powders (5-9). However, x-ray data indicate that the mobility of atoms near enzymes active sites is higher than average (10, 11) and, thus, the dielectric response should be enhanced. Furthermore, active sites are typically situated within several angstroms of the protein surface. A wide range of dielectric response was predicted from molecular simulations for various proteins: A gradual increase from Ϸ 3 in the center of globule to about Ϸ 10 at its periphery was found in refs. 12-19. Even higher values of 25-35 were calculated by taking into account fluctuations of ionizable surface groups (18,19). Finally, highly polarizable aqueous surroundings may contribute to the dielectric response, to increase the uncertainty even further.Thus, local dielectric properties at enzyme active sites have remained unclear. In the present paper, we attempt to resolve this issue by direct measurement. A spectroscopic technique proposed in refs. 20 and 21 and further developed in refs. 22-24 is used. The reorganization energy is determined from absorption and emission spectra of proflavine noncovalently bound in the active site of ␣-chymotrypsin. We evaluate the contributions to the dielectric reorganization of the protein interior and of the surrounding solvent by comparing the spectra of t...

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Section: Discussionsupporting

confidence: 69%

Low dielectric response in enzyme active site

Mertz

Krishtalik

2000

Proc. Natl. Acad. Sci. U.S.A.

125

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“…For charged groups, on the other hand, solvation energies are large, and the nonbonded parameters are more crucial for energetics. Therefore we have employed parameters for the negatively charged oxygens that have been carefully calibrated earlier against hydration free energies (17). The other van der Waals' parameters (16) pertaining to charged groups (e.g.…”

Section: Methodsmentioning

confidence: 99%

“…1. Each of these diabatic states (⌿ 1 -⌿ 5 ) is represented by an analytical force field of the standard molecular mechanics type (16,17). In the cases where standard force field parameters (16), viz.…”

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confidence: 99%

Computer Simulation of the Triosephosphate Isomerase Catalyzed Reaction

Åqvist

Fothergill²

1996

Journal of Biological Chemistry

Self Cite

103

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A major challenge for theoretical simulation methods is the calculation of enzymic reaction rates directly from the three-dimensional protein structure together with some idea of the chemical reaction mechanism. Here, we report the evaluation of a complete free energy profile for all the elementary steps of the triosephosphate isomerase catalyzed reaction using such an approach. The results are compatible with available experimental data and also suggest which of the possible reaction intermediates is kinetically observable. In addition to previously identified catalytic residues, the simulations show that a crystallographically observed active site water molecule plays an important role during catalysis and an intersubunit interaction that could explain the low activity of the monomeric enzyme is also observed. The calculations clearly demonstrate the important catalytic effects associated with stabilization of charged high energy intermediates and reduction of reorganization energy, which are likely to be general principles of enzyme catalyzed charge transfer and separation reactions.

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“…Both the simulation of l-arginine G1 and l-citrulline G1 free-states included 787 SPC model water molecules. The systems were assigned with modified GROMOS force field [22,23]. The l-arginine molecular fragment library file was created and a different set of partial atomic charges was assigned (Fig.…”

Section: Methodsmentioning

confidence: 99%