The Regulation of Malate Dehydrogenase by Citrate

Abstract

Malate Dehydrogenase (MDH) catalyzes the oxidation of malate to oxaloacetate through the reduction of NAD+ to NADH and is vital in a variety of cell processes important for metabolic and energy pathways. Glyoxasomal MDH (gMDH) is a homodimer with each subunit contains a dinucleotide binding domain and a malate binding domain. Citrate has long been suggested as an allosteric regulator of the enzyme but crystal structures show only citrate binding to the active site. To define the effects of citrate both kinetic and biophysical approaches have been used. The ability of citrate to inhibit gMDH is shown to be pH dependent in the range from pH 6‐8. At pH 7 & 8 Citrate only inhibits about 60% of the activity at apparently saturating concentrations suggesting subunit interactions are involved in the citrate effect. The extent of inhibition is also dependent upon substrate inhibition by oxaloacetate. Isothermal titration calorimetry (ITC) studies are also done to investigate the affinity of citrate for the active site of the dimeric gMDH. The effects of citrate on local flexibility and stability of subunit interactions [studied by limited proteolysis and dynamic light scattering] are correlated with the kinetic effects.This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB