Is a “Proton Wire” Concerted or Stepwise? A Model Study of Proton Transfer in Carbonic Anhydrase

Abstract

The energetics of proton transfer reactions in carbonic anhydrase (CA) have been studied with an active site model. Specifically, proton transfer from a zinc-bound water molecule to a histidine residue mediated by a numbers of water molecules was investigated. With two or three bridging water molecules, the proton transfers are fully or nearly fully concerted and only one saddle point exists. With an additional water molecule that forms a ring bridge, an intermediate is formed in which one of the water molecules exists as a hydronium ion. In contrast to previous calculations in which either a low-level of theory was employed or a stepwise mechanism was assumed, the energetics obtained from the current work are approximately consistent with the experimental estimates. In all of the scenarios, the motion of more than one proton is involved in the transition state, which is in agreement with the experimental observation that the reaction rates in H 2 O/D 2 O mixture have an exponential dependence on the fraction of D 2 O in the solvent. For three (W3) or four waters (W4), the proton transfer to the "His 64" model is hardly involved in the transition state, suggesting that the orientation of the proton acceptor is less important than for only two waters (W2). Thus, the W3 and W4 results are consistent with the experimental observation that many kinetic properties of the H64A mutant of CA in well-buffered imidazole solution are similar to the wild type. The barrier height increases, and the barrier frequency (and therefore, the contribution of tunneling) decreases as the number of bridging water molecules increases. Overall, these investigations demonstrate that the proton transfer reaction in CA is sensitive to the nature and structure of the water bridge, which would be influenced by the dynamics of the water molecules and amino acids in the active site of the protein.

Active-site solvent replenishment observed during human carbonic anhydrase II catalysis

“…The general base (B) for the proton transfer is likely to be mediated by ordered waters and His64 within the enzyme, where the hydrophilic side of the active site forms the hydrogen-bonded water network (W1, W2, W2 0 , W3a and W3b) that connects W Zn to His64. This hydrogen-bonded network is believed to act as a proton wire that reduces the work required to transfer a proton from W Zn to the bulk solvent for the regeneration of the zinc-bound OH À (2) Steiner et al, 1975;Cui & Karplus, 2003;Zheng et al, 2008;Fisher, Maupin et al, 2007;Silverman et al, 1979). Neutron studies have been utilized to observe the protonation states and orientation of water molecules in proteins (Langan et al, 2008).…”

Proton transfer through the water gossamer

“…In this spirit, Eigen (20) suggested that the proton could delocalize over extended hydrogen-bonded wires. There is evidence that this behavior can occur when water molecules form isolated chains, in confined environments like proteins and nanotubes (21)(22)(23). In addition, several spectroscopic experiments examining acid-base reactions in ice and water have suggested that fast PT occurs through the formation of transient water wires (24,25).…”

Tracking solvent and protein movement during CO 2 release in carbonic anhydrase II crystals

“…The structural examination of hCA II at near atomic resolution revealed that the solvent molecule W2 (the only ordered solvent molecule in the active site stabilized exclusively by other solvent molecules) is trigonally coordinated with equal distance (2.75 ± 0.02 Å) by W1, W3a, and W3b (16). Within this cluster of Significance Carbonic anhydrases catalyze the fast interconversion of carbon dioxide and water into bicarbonate and proton.…”