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Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site‐Directed Mutagenesis
Author(s) -
Carbini Luis A.,
Hersh Louis B.
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb03561.x
Subject(s) - choline acetyltransferase , mutant , histidine , biochemistry , active site , site directed mutagenesis , glutamine , chemistry , amino acid , enzyme , mutagenesis , acetyltransferase , choline , binding site , stereochemistry , microbiology and biotechnology , biology , acetylcholine , acetylation , gene , endocrinology
The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl‐CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila , pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His 268 , His 393 , and His 426 . Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His 268 Leu‐ChAT, His Z68 Gln‐ChAT, and His 393 Gln‐ChAT and inactive His 393 Leu‐ChAT, His 426 Leu‐ ChAT, and His 426 Gln‐ChAT. The kinetic constants K m(CoA) , K m(acetyloholine) . and V max were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His 393 Leu‐ChAT is disrupted but conserved in His 426 Leu‐ChAT and His 426 Gln‐ ChAT. These results suggest that His 426 is an essential catalytic residue and could serve as an acid/base catalyst.