Introduction of histidine analogs leads to enhanced proton transfer in carbonic anhydrase V.
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.
Earnhardt
J N
JN
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida, 32610-0267, USA.
Wright
S K
SK
Qian
M
M
Tu
C
C
Laipis
P J
PJ
Viola
R E
RE
Silverman
D N
DN
D000818:Animals; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D011522:Protons; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D011522:Protons; D006639:Histidine; D002256:Carbonic Anhydrases; D003545:Cysteine