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The Role of Methionine Residues in Activity and Subunit Communication in Malate Dehydrogenase
Author(s) -
Billue Farren,
Bell Ellis
Publication year - 2010
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.24.1_supplement.835.2
Subject(s) - protein subunit , malate dehydrogenase , biochemistry , chemistry , citrate synthase , dimer , enzyme , amino acid , methionine , active site , stereochemistry , organic chemistry , gene
Malate Dehydrogenase is a dimeric enzyme that catalyzes the oxidation of malate to oxaloacetate in a reaction involving both a hydride transfer [to NAD] and a proton abstraction. The enzyme demonstrates kinetic evidence for the involvement of subunit interactions in the catalytic activity and regulation [by citrate] but little is know concerning the mechanism of communication between the two active sites in the dimer. The subunit interface contains three conserved methionine residues [M62, M64 & M91] that are in close proximity to residues from the opposing subunit [P59, L63, Y273 & I88 as well as between M66 on one subunit and M62 & M91 on the opposing subunit]. These residues together with a Methionine close to the active site [M128] have been mutated using QuikChange mutagenesis to a variety of different amino acids to test their role in subunit communication and catalytic activity. The his‐tagged proteins are expressed, purified using Ni‐NTA affinity chromatography and characterized by MALDI‐tof and Circular Dichroism. The strength of subunit interactions is assessed using guanidine hydrochloride dissociation followed by dynamic light scattering. Initial rate kinetic studies demonstrate changes in both Vmax and Km of various mutants as well as indicating changes in citrate regulation of the protein. This work is supported by NSF Grant MCB 0448905 to EB and an HHMI summer stipend to FB