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Murine Carbonic Anhydrase V
Background:
The mammalian
carbonic anhydrases are a group of at least seven distinct zinc-containing
isozymes that catalyze the hydration of CO2. Carbonic anhydrase
V (CA V) is found primarily in the mitochondria of mammalian liver where it
is suggested to provide bicarbonate as a substrate for enzymes in the
pathways of both gluconeogenesis and ureagenesis. The overall maximal
velocity of catalysis of CO2 hydration is limited by
intramolecular proton transfer from the zinc-bound water that proceeds
through intervening water bridges (Silverman and Lindskog, 1988). However,
a significant difference between the isozymes is that CA V lacks a proton
shuttle mechanism that is as efficient as that of His 64 in the most
efficient isozyme, CA II. CA V has a tyrosine at position 64 and its
replacement by site-directed mutagenesis has shown that it does not play a
significant role in catalysis (Heck et al., 1996).
Results:
We have investigated
two sites for chemical modulation in murine CA, where our aim is to use
this approach to introduce unnatural amino acids that will provide an
internal proton transfer group to enhance catalysis. Reaction of an
introduced cysteine at position 91 did not lead to an enhancement of
activity with any of the histidine analog reagents. The selective
modification of an introduced cysteine at position 131 with
4-bromoethylimidazole and 4-chloromethylimidazole does result in
enhancements of up to three-fold, consistent with the introduction of a
functional proton transfer group into the active site. However, moving the
position of side chain attachment from carbon-4 to carbon-2 (modification
by 2-chloromethylimidazole) is a sufficient perturbation to prevent the
imidazole ring from occupying a suitable position for significant proton
transfer with the zinc-bound hydroxide (Earnhardt,
Wright et al.). This is the first example of the incorporation of an
unnatural amino acid analog of histidine to act as a proton shuttle group
in an enzyme.
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