Open AccessHydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme
Author(s) -
Rui He,
Judith R. Cristobal,
Naiji Jabin Gong,
John P. Richard
Publication year - 2020
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00801
Subject(s) - enzyme kinetics , dhap , protonation , chemistry , dihydroxyacetone phosphate , stereochemistry , dehydrogenase , active site , medicinal chemistry , catalysis , enzyme , biochemistry , organic chemistry , ion
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.