Premium
The Effects of Citrate on Glyoxasomal Malate Dehydrogenase
Author(s) -
Mays Jacqunae,
Marion James,
Bell Ellis
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.558.3
Subject(s) - protein quaternary structure , malate dehydrogenase , allosteric regulation , protein subunit , biophysics , biochemistry , chemistry , active site , citric acid cycle , proteolysis , citrate synthase , dehydrogenase , mutagenesis , enzyme , stereochemistry , biology , mutant , gene
Malate Dehydrogenase plays critical roles in the cytosol, the mitochondrion and in plants also in glyoxysomes. Mitochondrial and gyloxysomal forms of malate dehydrogenase are thought to be subject to allosteric regulation by citrate. To explore the mechanisms of citrate effects on the enzyme we have used a combination of initial rate kinetics, site directed mutagenesis, circular dichroism (in conjunction with thermal melts) to follow effects on secondary and tertiary structure, dynamic light scattering to follow effects on quaternary structure and limited proteolysis to explore local flexibility/accessibility changes. Citrate, which binds to only one of the two otherwise identical active sites, is a partial inhibitor under all circumstances examined (varied pH, forward or reverse reaction), has no effect on the quaternary structure of the protein but induces changes in local flexibility/accessibility in a loop near the subunit interface. Several mutations at the subunit interface (V195A, L269A, E256Q, H90Q)also impact citrate inhibition. Together these results suggest that citrate exerts its effects on activity by binding to the “empty” active site in a reciprocating subunit mechanism, eliciting altered subunit interactions that contribute to overall catalysis. This work is supported by NSF Grant MCB 0448905 to EB