Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site-Directed Mutagenesis

Abstract: 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 His²⁶⁸, His³⁹³, and His⁴²⁶. 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 His²⁶⁸Leu-ChAT, His^Z68Gln-ChAT, and His³⁹³Gln-ChAT and inactive His³⁹³Leu-ChAT, His⁴²⁶Leu- ChAT, and His⁴²⁶Gln-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 His³⁹³Leu-ChAT is disrupted but conserved in His⁴²⁶Leu-ChAT and His⁴²⁶Gln- ChAT. These results suggest that His⁴²⁶ is an essential catalytic residue and could serve as an acid/base catalyst.