The structure of bovine liver glutamate dehydrogenase: Insights into the mechanism of allosteric regulation

Peter Erik Peterson, Purdue University

Abstract

Bovine liver glutamate dehydrogenase (GDH) is a homohexameric, mitochondrial matrix enzyme that catalyzes the reversible oxidative deamination of L-glutamate to yield α-ketoglutarate and ammonia. This reaction requires the concomitant reduction of one molecule of NAD+ or NADP+ and can use either coenzyme with similar efficacy. GDH is a key link between catabolic and biosynthetic pathways and is found ubiquitously in both higher and lower organisms. Bovine liver GDH, unlike GDH from more primitive organisms, exhibits complex homotropic and heterotropic allosteric regulation including negative cooperativity and regulation by a large number of allosteric effectors. The largest difference between bovine and bacterial enzymes is a 48-residue insert in the C-terminal segment of the bovine liver sequence. The structure of bovine liver GDH crystallized as an abortive complex with L-glutamate, NADH, and the allosteric inhibitor, GTP, has been determined to 2.8Å. Five bound ligands were located: NADH and L-glutamate within the active site, a second NADH inside the hexamer core, and two GTPs near the hinge region connecting the two domains of the monomer. The core structure of bovine GDH is quite similar to previously reported bacterial structures. However, bovine GDH has additional “antennae” structures protruding from each trimer as right-handed helical bundles along the threefold axis. We propose that the antennae serve as communication conduits through which intersubunit allosteric regulation is either affected or enhanced. Crystal contacts suggest that the antennae also play a role in hexamer-hexamer interactions in solution and, perhaps, with other enzymes within the mitochodrial matrix. Comparison of the bovine liver and Clostridium symbiosum structures lead to the proposal that, within each subunit, enzyme regulation is affected via induced changes in the mobility of the NADH binding domain. Mutations in the antennae and GTP binding regions of human GDH are reported to cause a congenital syndrome characterized by hyperinsulinism and hyperammonemia. Since some of the identified mutations lie within the antennae structure, this syndrome lends further credence to the proposed role of the antennae in intersubunit communication. Furthermore, this syndrome suggests that GDH plays a significant role in the control of both insulin release and ammonia homeostasis.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Biophysics|Biochemistry

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