IDENTIFICATION OF AN ACTIVE-SITE ARGININE IN RAT CHOLINE-ACETYLTRANSFERASE BY ALANINE SCANNING MUTAGENESIS

Kinetic as well as chemical modification studies have implicated the p resence of an active site arginine in choline acetyltransferase, whose function is to stabilize coenzyme binding by interacting with the 5'- phosphate of the coenzyme A substrate. In order to identify this resid ue seven conserved arginines in rat choline acetyltransferase were con verted to alanine by site-directed mutagenesis, and the properties of these mutants were compared with the wild type enzyme. Substitution of arginine 452 with alanine resulted in a 7-12-fold increase in the K-m for both CoA and acetylcholine as well as K-cat with little change in the K-m for dephospho-CoA. Product inhibition studies showed choline to be a competitive inhibitor with respect to acetylcholine, indicatin g R452A follows the same Theorell-Chance kinetic mechanism as the wild type enzyme. Similar results were obtained with R452Q and R452E, with the latter showing the largest changes in kinetic parameters, These f indings are consistent with Arg-452 mutations increasing the rate cons tant, k(5), for dissociation of the coenzyme from the enzyme. Direct e vidence that arginine 452 is involved in coenzyme A binding was obtain ed by showing a 5-10-fold decrease in affinity of the R452A mutant for coenzyme A as determined by the ability to protect against phenylglyo xal inactivation as well as thermal inactivation.