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The Varied Roles of Conserved Arginine Residues near Beta‐turns in Glyoxysomal Malate Dehydrogenase
Author(s) -
Hedrick Shan,
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.469.3
Subject(s) - arginine , malate dehydrogenase , amino acid , glutamine , biochemistry , mutant , chemistry , enzyme , biophysics , biology , gene
In previous experiments, poly‐glutamine insertion mutants, Q301 and Q240, at pre‐existing glutamines within or very near beta turns we made. The crystal structure of Q301 indicated minimal overall changes. Q240 interacted with other proteins to form aggregates. We decided to investigate the roles of beta turns. Analysis of temperature factors of gMDH indicated a flexible beta turn. Within this “hot‐spot” flexible loop, lies R130 and R124, part of the malate binding site, which also includes R196. These arginines and R185 were mutated to a variety of amino acids. Arginine to glutamate mutations showed a large reduction in activity, however NADH was bound more tightly and oxaloacetate more weakly. R130Q and R130L had activities similar to native with R130L showing enhanced activity. R130Q and R130L also had similar CD spectra. R130E, however, had a significantly altered spectrum suggesting increased beta‐strand formation. Thermal denaturation studies indicated that R130Q had similar stability to native, but R130E had significantly lower stability. R130L, however, may be more stable due to the hydrophobicity of the mutation. We conclude that various arginine residues play roles both in substrate binding and catalysis. This work is supported by NSF Grant MCB 0448905 to EB