Hydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme

[Image: see text] K120 of glycerol 3-phosphate dehydrogenase (GPDH) lies close to the carbonyl group of the bound dihydroxyacetone phosphate (DHAP) dianion. pH rate (pH 4.6–9.0) profiles are reported for k(cat) and (k(cat)/K(m))(dianion) for wild type and K120A GPDH-catalyzed reduction of DHAP by NADH, and for (k(cat)/K(d)K(am)) for activation of the variant-catalyzed reduction by CH(3)CH(2)NH(3)(+), where K(am) and K(d) are apparent dissociation constants for CH(3)CH(2)NH(3)(+) and DHAP, respectively. These profiles provide evidence that the K120 side chain cation, which is stabilized by an ion-pairing interaction with the D260 side chain, remains protonated between pH 4.6 and 9.0. The profiles for wild type and K120A variant GPDH show downward breaks at a similar pH value (7.6) that are attributed to protonation of the K204 side chain, which also lies close to the substrate carbonyl oxygen. The pH profiles for (k(cat)/K(m))(dianion) and (k(cat)/K(d)K(am)) for the K120A variant show that the monoprotonated form of the variant is activated for catalysis by CH(3)CH(2)NH(3)(+) but has no detectable activity, compared to the diprotonated variant, for unactivated reduction of DHAP. The pH profile for k(cat) shows that the monoprotonated K120A variant is active toward reduction of enzyme-bound DHAP, because of activation by a ligand-driven conformational change. Upward breaks in the pH profiles for k(cat) and (k(cat)/K(m))(dianion) for K120A GPDH are attributed to protonation of D260. These breaks are consistent with the functional replacement of K120 by D260, and a plasticity in the catalytic roles of the active site side chains.