1P091
Theoretical Study of a -stacking Interaction Effect to the Orientationof His64 of Carbonic Anhydrase(Kanazawa University*, Japan Advanced Institute of Technology**)o Muhamad Koyimatu*, Hideto Shimahara**, Kimikazu Sugimori*, Tetsuya
Sugimori*, Kazutomo Kawaguchi*, Hiroaki Saito*, Hidemi Nagao*[Introduction]
Carbonic Anhydrase (CA) is zinc-containing enyme that catalyses the reversible hydration ofcarbon dioxide to form bicarbonate and excess proton.
CO2 + H2O ⇋ HCO3- + H+
Human Carbonic Anhydrase II (HCA II) has the fastest value among those of CA isozymes.
Histidine at position 64 is accepted to facilitate the transfer of the productive proton from thezinc-bound water to a buffer molecule in bulk-water through intervening hydrogen bonded
water molecules.
The final step of proton transfer has been assumed to be connected with a rotational orswinging motion of the side chain of His64 because this residue has two conformations, “in”
and “out”. The properties of the imidazole side chain of His64 should be tuned by Trp5
because the indole ring is located near to imidazole ring of His64 (~4 Å), in which the πstacking interaction can occur in such aromatic rings. According to the crystal structure, theindole ring of Trp5 planar parallel to the imidazole ring of the “out” conformation of His64 is
an off-centered structure, in which a face-to-face of -stacking interaction should be formed
to stabilize the two aromatic rings.1 A researcher reported that His64 has a potential to berotated in the catalysis by using molecular dynamic simulations.2 However, the indole ring of
Trp5 has not been considered in the model system.
In order to investigate the possibility that the indole ring of Trp5 interrupts the rotationalmotion of His64, we constructed the His64 and Trp5-containing model, and estimate theeffect of the -stacking interaction energy.
[Experiment]
We constructed a model system that consists of His64
and Trp5 (Figure 1). The coordinates were obtained
from the protein database (PDB) file of the crystal
structure of HCA II (2CBA). The 1 angle of His64
was adjusted manually to clearly see the interactionenergy between His64 and Trp5. The pKa value for theimidazole ring is approximately 7.0, both the acid andbase forms are present. The acid form is imidazolium

Figure 1.The model system included His64 andTrp5.

ion, and the base form is the N1-H tautomer and the N2-H tautomer. Considering that, we
simulated all three forms of imidazole.
The density-functional theory (DFT) method was employed to optimize the position ofhydrogen atoms in model system. The position of hetero atoms (C, N, and O) was fixed.
Considering electron-electron interaction, DFT method was not enough to estimate the stacking interaction. The second-order Moller-Plesset perturbation theory applied to theoptimized structure from MP2 calculation.
[Results and Discussion]
The calculated energy values were plotted as a function of the 1 angle of the His64 toinvestigate the profile of the -stacking interaction. We superimposed two curves of theenergy data at the biggest 1 angle value. There was expected to be the lowest -stacking
interaction at this angle (Figure 2). The -stacking interaction occurs at the 1 angle around 50°. At this 1 angle, His64 form a face-to-face parallel interaction with Trp5. The curve ofthree types of imidazole shows that addition Trp5 to the model system could stabilize thestructure. The imidazolium form preferred the “out” conformation, similar with the modelsystem reported by Maupin.1 However Maupin only reported one curve in tautomeric form,probably the N1-H tautomer, because it preferred “in” conformation. In our result, the N2H tautomer preferred the “out” conformation. In our result, the N1-H tautomer preferred the“in” conformation, while the N2-H tautomer preferred the “out” conformation. However
Maupin only reported one curve in tautomeric form, probably the N1-H tautomer, because it
preferred “in” conformation. The different of preferred orientation of two tautomers could
support the tautomerism of His64 to the proton transfer mechanism.3
The addition of Trp5 also increases the energy barrier that could restrict the rotational motionof His64. We need more investigation to verify the effect of -stacking interaction in thecatalytic mechanism of HCA II.

Figure 2. The energy profile for each types of imidazole of His64.

1. D.N. Silverman and R. McKenna: Acc. Chem. Res. 40 (2007) 669.
2. C. Maupin and G.A. Voth: Biochemistry. 46 (2007) 2938.
3. H. Shimahara, et al.: J. Biol. Chem. 282 (2007) 9646.