Glutamate and Aspartate as Proton Shuttles in Mutants of Carbonic Anhydrase

Abstract

Maximal turnover rates for the hydration of CO2 and the depletion of 18O from CO2 catalyzed by carbonic anhydrase III (CA III) and carbonic anhydrase V (CA V) are limited by proton transfer involving zinc-bound water or hydroxide in the active site. We have investigated the capacity of glutamic and aspartic acids at position 64 in human CA III and murine CA V to act as proton shuttles in this pathway. The distance from the Calpha of position 64 to the zinc is near 9.5 A in the crystal structures of both CA III and CA V. Rates of intramolecular proton transfer between these proton shuttle groups and the zinc-bound water molecule were estimated as the predominant rate-contributing step in the catalytic turnover kcat in the hydration of CO2 measured by stopped flow and in the 18O exchange between CO2 and water measured by mass spectrometry. We found that both glutamate and aspartate residues at position 64 are efficient proton shuttles in HCA III. The rate constant for intramolecular proton transfer from either residue to zinc-bound hydroxide is 4 x 10(4) s-1, about 20-fold greater than that of the wild type which has lysine at position 64. When the active site residue Phe 198 in human CA III was replaced with Leu, measurement of catalysis showed that Glu 64 retained but Asp 64 lost its capacity to act as a proton shuttle. These observations were supported in studies of catalysis by murine CA V which contains Leu 198; here again, Glu 64 acted as a proton shuttle, but Asp 64 did not. Phe 198 in HCA III is thus a significant factor in the capacity of the active site to sustain proton transfer, possibly through its stabilization of hydrogen-bonded water bridges that enhance proton translocation from both Glu and Asp at position 64 to the zinc-bound hydroxide.

Chemical Rescue in Catalysis by Human Carbonic Anhydrases II and III

“…Table 2 gives the resulting values of the intrinsic kinetic barrier ∆G q 0 and the work function or thermodynamic contribution w r (dehydration direction) to the proton transfer (17,34,35). 2 These data demonstrate a general result that has also been observed for intramolecular proton transfer in HCA III (17,36,37) and for intermolecular proton transfer involving exogenous donors in CA V (33); in this application of Marcus theory to proton transfer in carbonic anhydrase the intrinsic barrier ∆G q 0 is small, less than 2 kcal/mol, and the work functions are large, generally 6-11 kcal/mol ( Table 2). The data for H64A HCA II obtained here are qualitatively similar to those of the previous studies in Table 2.…”

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

“…∼5 (Jewell et al, 1991;Qian et al, 1997) <6 (Elder et al, 2007) Escherichia coli CA IV (Mickevičiūtė et al, 2017) 6.2 (Baird et al, 1997) (Innocenti et al, 2009) CA IX (full length) 6.49 (Innocenti et al, 2009) CA XII (Jogaitė et al, 2013) A database of intrinsic enthalpies and entropies of sulfonamide inhibitor binding to all 12 catalytically active human CAs…”

Voltage-Gated Proton Channels: Molecular Biology, Physiology, and Pathophysiology of the HVFamily

“…Replacing Lys 64 in CA-III with His increases the catalytic rate severalfold, as does simple addition of imidazole buffer (241). Intriguingly, replacing Lys 64 in CA-III with either Asp or Glu increased the internal proton transfer rate 20-fold (411), indicating that in this enzyme, Asp and Glu transfer protons even more efficiently than His. However, despite its considerable facilitation of proton transfer, His 64 does not act as a selectivity filter.…”