GLUTAMATE-190 IS A GENERAL ACID CATALYST IN THE 6-PHOSPHOGLUCONATE-DEHYDROGENASE-CATALYZED REACTION

Site-directed mutagenesis was used to change E190 of sheep liver 6-pho sphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possi ble role as a general acid catalyst. Each of the mutant proteins was c haracterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in p H-rate profiles with no observable pK on the basic side of the V/K-6PG profile. Mutations that change the pK of the group at position 190 re sult in the expected pK perturbations in the V/K-6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are cons istent with a rate-limiting tautomerization of the 1,2-enediol of ribu lose 5-phosphate consistent with the proposed role of E190. Data are a lso consistent with some participation of E190 in an isomerization req uired to form the active Michaelis complex.