W0125

Structure of a Glutamate Dehydrogenase Abortive Complex from Bovine Liver. Peter E. Peterson and Thomas J. Smith, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA

The structure of bovine liver glutamate (GDH) crystallized as an abortive complex with substrate glutamic acid, coenzyme NADH, and inhibitor GTP has been determined at 2.8Å resolution. Bovine liver GDH is a hexamer of identical subunits with a total molecular weight of 336 kDa and lies at a key junction between carbon and nitrogen metabolism. Mammalian GDH catalyzes the reversible oxidative deamination of glutamate to yield [alpha]-ketoglutarate and ammonia using either NAD⁺ or NADP⁺ as coenzyme with similar efficacy.

Bovine liver GDH undergoes a concentration-dependent aggregation which is independent of enzymatic activity. This aggregation has hindered previous crystallization attempts. Mammalian GDH exhibits complex allosteric behavior including allosteric regulation by purine nucleotides and negatively cooperative binding of coenzyme in the presence of glutamate. Structures have been determined for several bacterial forms of GDH, but neither the complex allosteric behavior nor the aggregative tendencies are observed in these enzymes. Relatively low sequence identity exists between the bovine liver and bacterial forms of GDH with the most significant difference residing in a 48-residue insert at residue 395 in the bovine sequence.

The structure of bovine liver GDH has been solved by MIR employing two heavy atom derivatives. The hexamer of GDH exhibits 32 symmetry and domain structures similar to the bacterial structures. The major difference exists in the presence of the 48-residue insert which forms an "antenna"-like structure extending along the 3-fold axis. In the hexamer, these "antenna" interact with each other forming helix bundles on either side of the hexamer. This antenna-bundle may be the origin of the complex allosteric behavior and aggregative tendencies of bovine liver GDH.

Biochemical studies have already demonstrated that modification of K420, which resides at the tip of the antenna, results in a decrease in the ability of the enzyme to aggregate. It has also been shown that GTP causes a similar decrease in aggregation. Unassigned density has been located at the base of the antenna near Y262 which we believe to be GTP. Studies with GTP analogs have identified Y262 as one of the possible binding sites for GTP.