Proton transfer in catalysis and the role of proton shuttles in carbonic anhydrase

Abstract

The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the alpha class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pK(a) for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pK(a) between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in DeltapK(a); however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase.

Figure 1

The active site of HCA II in a ball-and-stick diagram taken from ref [15]. The zinc ion and the oxygen molecules of water molecules are shown as black and red spheres, respectively. The water network of the active-site is labeled W1, W2, etc. and W_DW is the deep water. Hydrogen bonds involving solvent molecules are represented as dashed red lines. The dual conformation of the His64 side chain is shown in both inward and outward conformations.

Figure 2

Free energy plot of the logarithm of k_B (s⁻¹) versus ΔpK_a (pK_{a ZnH2O} – pKa _His64) for (filled symbols) the wild type and the mutants of HCA II identified by number in Table 2; and (open circles) for H64H HCA II with proton transfer provided predominantly by derivatives of imidazole and pyridine acting as exogenous proton donors with the solid line a best fit of Marcus Rate Theory (Figure 5 from ref [24]). Data were obtained by ¹⁸O exchange at 25 °C.