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The Roles of Conserved Cysteine Residues in the Structure and Function of Glyoxysomal Malate Dehydrogenase
Author(s) -
Jones Rachel,
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.581.9
Subject(s) - malate dehydrogenase , biochemistry , cysteine , mutant , protein tertiary structure , enzyme , biology , dehydrogenase , conserved sequence , cooperativity , subcellular localization , dtnb , function (biology) , chemistry , microbiology and biotechnology , peptide sequence , cytoplasm , glutathione , gene
Bioinformatics analysis of malate dehydrogenases from both eukaryotic and prokaryotic sources indicate the existence of a number of cysteine residues that are highly conserved in mitochondrial and glyoxysomal forms of the enzyme (C282 and C295) as well as a cluster of cysteines (145, 151, 152) that are not conserved. In order to probe the roles of these conserved cysteines residues in both structure and folding of the enzyme as well as the catalytic activity, a number of site directed mutants (C145V‐C151S‐C152S triple mutant, C282S and L, and C295S) of watermelon glyoxysomal malate dehydrogenase have been constructed, expressed, purified and characterized. The resultant mutants are characterized for reactivity to DTNB (native protein reacts slowly indicating that one or more of the SH groups are somewhat accessible), secondary and tertiary structure using CD and CD/Thermal melts, and catalytic activity. Preliminary results indicate that all mutants have significant activity with differences in Km and Vmax. Similarly all mutants show evidence via CD for secondary structure but show some differences in stability and cooperativity of folding suggesting that none of the conserved cysteines are critical for activity but contribute to overall structure and function. This work is supported by NSF Grant MCB 0448905 to EB