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Title
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Introduction of histidine analogs leads to enhanced proton transfer in carbonic anhydrase V.
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Authors
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J.N.Earnhardt,
S.K.Wright,
M.Qian,
C.Tu,
P.J.Laipis,
R.E.Viola,
D.N.Silverman.
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Ref.
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Arch Biochem Biophys, 1999,
361,
264-270.
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PubMed id
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Abstract
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The rate-limiting step in the catalysis of the hydration of CO2 by carbonic
anhydrase involves transfer of protons between zinc-bound water and solution.
This proton transfer can be enhanced by proton shuttle residues within the
active-site cavity of the enzyme. We have used chemical modulation to provide
novel internal proton transfer groups that enhance catalysis by murine carbonic
anhydrase V (mCA V). This approach involves the site-directed mutation of a
targeted residue to a cysteine which is then subsequently reacted with an
imidazole analog containing an appropriately positioned leaving group. Compounds
examined include 4-bromoethylimidazole (4-BEI), 2-chloromethylimidazole (2-CMI),
4-chloromethylimidazole (4-CMI), and a triazole analog. Two sites in mCA V, Lys
91 and Tyr 131, located on the rim of the active-site cavity have been targeted
for the introduction of these imidazole analogs. Modification of the introduced
Cys 131 with 4-BEI and 4-CMI resulted in enhancements of up to threefold in
catalytic activity. The pH profiles indicate the presence of a new proton
shuttle residue of pKa near 5.8, consistent with the introduction of a
functional proton transfer group into the active site. This is the first example
of incorporation by chemical modification of an unnatural amino acid analog of
histidine that can act as a proton shuttle in an enzyme.
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