The Linkage Between Citrate, pH Regulation and Protein Folding in Glyoxysomal Malate Dehydrogenase

Abstract

Malate Dehydrogenase, a dimeric enzyme that catalyzes the NAD/NADH‐dependent reversible oxidation of malate into oxaloacetate, is involved in the Citric Acid and Glyoxylate Cycles as well as the malate‐aspartate shuttle. Citrate and pH appear to regulate the activity of glyoxysomal and mitochondrial MDH. The aim of this project is to study the linkage between pH and citrate regulation of Malate Dehydrogenase and protein stability and folding cooperativity. Over the pH range from 6.0 to 8.0 clear effects on secondary structure as shown by circular dichroism are seen. Thermal melts using CD at 222nm with data fitting via a four parameter Hill equation to give both Tm and a cooperativity parameter indicate effects on the unfolding cooperativity as well as the overall stability. Likewise, Citrate binding at various pH values clearly affects both, suggesting a linkage between secondary and tertiary structure stabilities, allosteric behavior and folding cooperativity.This work is supported by NSF Grant MCB 0448905 to EB.