title: Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study.
abstract: The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324, USA.
authors.forename: Edgar
authors.initials: E
authors.lastname: Deu
authors.forename: Jack F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D000494:Allosteric Regulation; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D055503:Protein Multimerization; D000637:Transaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014444:Tyrosine Transaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000637:Transaminases; D001219:Aspartate Aminotransferases; D014444:Tyrosine Transaminase