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Allosteric Regulation of Glyoxasomal Malate Dehydrogenase by Citrate Involves Reciprocating Active Site Communication.
Author(s) -
Mays Jacqunae,
Bell Ellis
Publication year - 2013
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.27.1_supplement.789.10
Subject(s) - allosteric regulation , protein quaternary structure , protein subunit , active site , malate dehydrogenase , biochemistry , citric acid cycle , biophysics , chemistry , biology , stereochemistry , enzyme , gene
Mitochondrial and glyoxysomal 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) and ANS Fluorescence 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, has no effect on quaternary structure but induces changes in local flexibility/accessibility in a loop near the subunit interface. Several mutations at the subunit interface (V195A, L269A, E256Q, H90Q) impact citrate inhibition. Studies of ANS binding have also been used to explore carboxylic acid induced changes in conformation (glyoxysomal, mitochondrial and cytoplasmic). The results suggest carboxylic acid substrates of MDH induce a reciprocating subunit mechanism that may contribute to catalysis, and citrate exerts its effects by binding to the “empty” active site in this reciprocating subunit mechanism, eliciting altered subunit interactions that contribute to regulation of the overall catalytic cycle. Supported by NSF Grant MCB 0448905 to EB