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Disruption of the active site solvent network in carbonic anhydrase II decreases the efficiency of proton transfer.

The importance of maintaining the active site water network for efficient proton transfer was investigated by substituting amino acids of varying size at position 65 in carbonic anhydrase II (including four amino acids found in other CA isozymes, F, L, S, and T, and two amino acids that do not occur naturally at position 65, G and H) and measuring the rate constants for the proton transfer reactions in the variant carbonic anhydrases. Intramolecular proton transfer between zinc-bound water and H64 is significantly inhibited by the introduction of bulky residues at position 65; kcat for CO2 hydration decreases up to 26-fold, comparable to the observed decrease in intramolecular proton transfer caused by removal of H64 [Tu, C., Silverman, D. N., Forsman, C., Jonsson, B.-H., & Lindskog, S. (1989) Biochemistry 28, 7913-7918]. Intermolecular proton transfer between protonated H64 and external buffer is also inhibited, although to a lesser degree. Furthermore, an alternative proton transfer pathway, consisting of an active site solvent-mediated proton transfer from zinc-water to imidazole buffer, is inhibited in the A65F, A65L, and A65H CAII variants. Therefore, the active solvent bridge between zinc-bound water and H64 is disrupted by substitutions at position 65. The inhibition of proton transfer reactions correlates with the disruption of the crystallographically observed solvent network in the CA active site and rotation of the proton acceptor, H64 [Scolnick, L. R., & Christianson, D. W. (1996) Biochemistry 35, 16429-16434], suggesting that this solvent network, including water molecules 292, 264, and 369, or a structurally related network, forms the proton transfer pathway in CAII for both intramolecular proton transfer and stimulation of proton transfer in imidazole buffers.