406
J . Am. Chem. SOC.1991, 113,406-41 1
C 0 2 Binding to Human Carbonic Anhydrase I1 Kenneth M. Men, Jr. Contribution from the Department of Chemistry and the Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802. Received February 15, 1990
Abstract: We report molecular dynamics and free energy perturbation simulations aimed at studying the binding of CO, with human carbonic anhydrase I1 (HCAII). We first describe a novel approach by which metal ions can be realistically incorporated into molecular mechanical force fields, by simply using electrostatic potential derived point charges. These charges better represent the active site environment and eliminate the large electrostatic interactions that result from the use of formal charges (e.g. +2 for zinc) for metal ions. From our molecular dynamics simulations we have identified two CO, binding sites. The first binding pocket is about 3-4 A away from the zinc ion (Zn-C distance) and is surrounded by Val 121,143,207,Trp 209, Leu 198, His 119, Thr 199, and the residue consisting of Zn-OH. The second pocket is about 5-6 A away from zinc and is formed by His 64,94,96, Ala 65, Asn 244, Tyr 7, Phe 93, and Thr 200. Further analysis of the MD trajectories yields information regarding the fluctuations of a CO, molecule bound in these two sites and the conformational flexibility of His 64. Using free energy perturbation techniques we have also evaluated the absolute free energies of binding for these two sites. The pocket closer to the zinc ion has a higher affinity for COzthan does the more remote site. The calculated binding energies are -3.37 i I . 14 and -2.17 f 0.83 kcal/mol, respectively. Our computed free energies of binding are in reasonable agreement with the experimental value (-2.2 kcal/mol), but importantly our computed values indicate that the closer of the two pockets has a higher affinity for CO,. These observations as well as the proximity of C 0 2 to the functionally important zinc ion suggest that the closer site is catalytically important, while the second site serves to increase the surface area by which a C 0 2molecule is recognized and bound by HCAII. By taking advantage of both molecular dynamics and free energy perturbation techniques new insights into the dynamics and energies of protein-substrate interactions can be readily obtained.
Introduction The precise way in which a substrate molecule interacts and then binds to its protein host is very difficult to characterke, but it is crucial to have this knowledge in order for us to extend our understanding of protein structure and function.' Kinetics has given us free energies, in the form of binding constants, for the association of an inhibitor or substrate molecule with a protein.' This allows us to build up an "energetic database" which provides us with information that correlates the structure (or a structural feature) of an inhibitor or substrate molecule with binding efficacy. This is turn gives us information regarding the disposition of residues in the active site of an enzyme. X-ray crystallography has provided us with numerous insights into protein-ligand interactions by providing us with information regarding the spatial interaction of an inhibitor or a substrate molecule with its protein host., Modern NMR techniques are providing us with this same sort of inf~rmation.~Furthermore, modern theoretical techniques, such as molecular dynamics (MD)4 and MD free energy perturbation (MDFEP) simulation^^,^ have started to provide us with both structural and energetic information regarding proteinsubstrate interactions. Herein, we present results from MD and MDFEP simulations that probe the noncovalent interaction of the substrate molecule CO2 with its protein host, human carbonic anhydrase I1 (HCAII). The combined use of these two types of simulations allows us to provide both qualitative structural and energetic insights into COz/HCAII interactions. HCAII is a small monomeric protein consisting of 260 residues with a total molecular weight of about 30 kd. The zinc ion in all known carbonic anhydrases is of catalytic importance.6 The ~
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( I ) Fersht, A. R. Enzyme Structure and Mechanism; W. H. Freeman and Co.: New York, 1985. (2) Blundell, T. L.; Johnson, L. N . Protein Crystallography; Academic Press: New York, 1976. (3) Braun, W. Q. Rev. Biophys. 1987, 19, 115. Wiithrich, K. N M R of Proteins and Nucleic Acids; Wiley: New York, 1986. (4) Brooks, C. L., 111; Karplus, M.: Pettitt, B. M. Proteins: A Theoretical Perspective of Dynamics, Structure, and Thermodynamics. Advances in Chemical Physics; John Wiley and Sons: New York, 1988; Vol. LXXI. McCammon, J . A.; Harvey, S.C. Dynamics oJProieins and Nucleic Acids; Cambridge University Press: New York, 1987. ( 5 ) van Gunsteren, W. F. Protein Eng. 1988,2, 5 . Mezei, M.; Beveridge, D. L. Ann. N.Y. Acad. Sci. 1986,482, 1. Jorgensen, W. L. Acc. Chem. Res. 1989, 22, 184. Kollman, P. A.; Merz, K. M., Jr. Arc. Chem. Res. 1990, 23, 246.
Scheme I R2n.OH
R3Zn-OH
\ R3Zn.OH
+ Con
2
R3Zn.HC03
coordination of the zinc ion is to three histidines, His 94,96,1 19.69 The fourth coordination site is occupied by a water molecule in the low-pH form of the enzyme (
