Structure-Based Design of an Intramolecular Proton Transfer Site in Murine Carbonic Anhydrase V

Abstract

Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton. The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme. Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity. The crystal structure of wild-type carbonic anhydrase V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc. We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry. Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer. However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65. This is the first structure-based design of an efficient proton transfer site in an enzyme.

Catalytic Properties of Murine Carbonic Anhydrase IV

“…6). A similar observation was made for murine CA V (33). Again, this feature was not observed in the 18 O-exchange experiment (Fig.…”

“…6). 4 One of these groups is His 64 acting as a proton shuttle, as described above, and the second is at least one other proton shuttle of pK a near 9 that has not yet been identified but that is similar to that observed in CA V (33). The activation of k cat through chemical rescue caused by the presence of 25 mM imidazole (Fig.…”

“…1). This Thr 65 may block the proton shuttle function of His 64 in a manner similar to that of Phe 65 in CA V (33). However, in CA II Jackman et al (34) found no effect of the replacement Ala 65 3 Thr on the function of the proton-shuttle mechanism involving His 64 .…”

Molecular dynamics simulations of human carbonic anhydrase II: Insight into experimental results and the role of solvation

“…Mutation of His64 with other residues under various conditions 16,23 has suggested that the proton transfer from the Zn-bound water to His64 is the pathway which leads to the high catalytic activity. This result has led to the first example of structure-based design of intramolecular proton transfer in CA V. 24,25 It is also interesting that the X-ray crystallographic studies 3 have found that the conformation of His64 is sensitive to the pH conditions. However, from the X-ray structural data alone, it was not clear as to the cause of the shift in the conformations.…”

Molecular Determinants ofS-Glutathionylation of Carbonic Anhydrase 3

“…Car1 and Car2 have a serine residue in place of Cys186, preventing glutathionylation. However, the mitochondrial Car5 has three surfaceexposed cysteine residues, including those corresponding to Cys181 and Cys186 in Car3 (12). We expect that mitochondrial Car5, like cytosolic Car3, will be particularly susceptible to glutathionylation.…”