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Interaction of Residues Aspartate 549 and Arginine 404 Modulates Active Site Accessibility and Intersubunit Communication in the E1 Subunit of Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex
Author(s) -
Kale Sachin,
Jordan Frank
Publication year - 2007
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.21.5.a645-a
Subject(s) - active site , protein subunit , chemistry , stereochemistry , enzyme kinetics , residue (chemistry) , pyruvate dehydrogenase complex , biophysics , conformational change , biochemistry , crystallography , enzyme , biology , gene
Dynamic active site inner and outer loops (spanning 401–413 and 541–557) in the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit undergo disorder to order transformation in the presence of an intermediate analogue (Arjunan et al., J.Biol.Chem . 2006). This activity is essential for catalysis by E1and for intersubunit communication with E2. Crystallographic studies indicate that H407, a multifunctional amino acid residue on the inner loop, is essential for ordering of both loops. D549 on the outer loop is within hydrogen bonding distance to inner loop residue N404. The D549A substitution resulted in decreased k cat (256 fold), reduction in intersubunit communication. 10‐fold slower predecarboxylation steps, only modestly affected K d for substrate binding. Viscosity‐dependant kinetics and the appearance of carboligase products suggest impaired loop closure over active site. These observations point to an important role of this residue in sequestering the active site from the reaction medium, and more importantly in intersubunit communication. The evidence also strongly points to this activity being a dynamic process. Supported by: NIH GM‐050380.