Glutamate and aspartate as proton shuttles in mutants of carbonic anhydrase

Maximal turnover rates for the hydration of CO2 and the depletion of O-18 from CO2 catalyzed by carbonic anhydrase UT (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 C alpha of position 64 to the zinc is near 9.5 Angstrom 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 k(cat) in the hydration of CO2 measured by stopped flow and in the O-18 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 PICA m. 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. Phr 198 in PICA 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.