Measuring the Oxidation State and Enzymatic Activity of Glyceraldehyde Phosphate Dehydrogenase (GAPDH).

Abstract

Glyceraldehyde phosphate dehydrogenase (GAPDH) is a highly conserved, essential, and abundant enzyme that catalyzes a rate-determining step of glycolysis. GAPDH catalyzes the nicotinamide adenine dinucleotide (NAD+)- and inorganic phosphate-dependent oxidation and phosphorylation of glyceraldehyde phosphate (GAP) to form 1,3-bisphosphoglycerate (BPG). As part of its mechanism of action, GAPDH employs a redox-sensitive cysteine that serves as a nucleophile to form a covalent adduct with GAP in order to set-up subsequent oxidation and phosphorylation steps. As a result of the redox sensitivity of the active site cysteine residue, GAPDH is susceptible to oxidative inactivation by oxidants such as hydrogen peroxide (H2O2). Indeed, numerous studies have demonstrated that oxidative inactivation of GAPDH has important metabolic consequences for adaptation to life in air and oxidative stress since decreased GAPDH activity results in the rerouting of carbon flux away from glycolysis and toward the pentose phosphate pathway to produce the key cellular reductant and antioxidant, NADPH. Thus, the ability to probe GAPDH oxidation and activity provides an important snapshot of the intracellular redox environment and glycolytic flux. Herein, we describe methods to measure reduced and oxidized GAPDH using thiol alkylation assays as well as GAPDH enzymatic activity.