Dissecting the Antenna in Human Glutamate Dehydrogenase: Understanding Its Role in Subunit Communication and Allosteric Regulation.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

Internal Motions of Basic Side Chains of the Antennapedia Homeodomain in the Free and DNA-Bound States.

Basic side chains play crucial roles in protein-DNA interactions. In this study, using NMR spectroscopy, we investigated the dynamics of Arg and Lys side chains of the fruit fly Antennapedia homeodomain in the free state and in the complex with target DNA. We measured 15N relaxation for Arg and Lys side chains at two magnetic fields, from which generalized order parameters for the cationic groups were determined. Mobility of the R5 side chain, which makes hydrogen bonds with a thymine base in the DNA minor groove, was greatly dampened. Several Lys and Arg side chains that form intermolecular ion pairs with DNA phosphates were found to retain high mobility with the order parameter being <0.6 in the DNA-bound state. Interestingly, some of the interfacial cationic groups in the complex were more mobile than in the free protein. The retained or enhanced mobility of the Arg and Lys side chains in the complex should mitigate the overall loss of conformational entropy in the protein-DNA association and allow dynamic molecular recognition.