Structures of Bovine Glutamate Dehydrogenase Complexes Elucidate the Mechanism of Purine Regulation Thomas J. Smith 1 *, Peter E. Peterson 1 , Timothy Schmidt 1 , Jie Fang 2 and Charles A. Stanley 2 1 Department of Biological Sciences, Purdue University West Lafayette, IN 47907, USA 2 The Children`s Hospital of Philadelphia, Endocrinology Division, Rm. 410D, 3516 Civic Center Blvd. Philadelphia PA 19104, USA Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of L-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable ef®- cacy and exhibits a complex pattern of allosteric inhibition by a wide var- iety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD  . The structures presented here have re®ned and modi®ed the previous struc- tural model of allosteric regulation inferred from the original boGDH  NADH  GLU  GTP complex. The boGDH  NAD   a-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the ``pivot helix'' of the NAD  binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH  NADPH  GLU  GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identi®ed. However, ADP, NAD  , and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD  , and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition. # 2001 Academic Press Keywords: allostery; glutamate dehydrogenase; purine regulation; hyperinsulinism *Corresponding author Introduction Glutamate dehydrogenase plays a pivotal role in nitrogen and carbon metabolism. 1 In the oxidative deamination reaction, GDH feeds the TCA cycle by converting L-glutamate to 2-oxoglutarate, whereas the reductive amination reaction may supply nitro- gen for several biosynthetic pathways. In yeast and most bacteria, separate forms of enzyme are made to utilize either NAD(H) or NADP(H) depending upon the metabolic needs of the organisms. 2 Some archaebacteria are the exception to this rule as the coenzyme V max /K m ratios differ by only about one order of magnitude. 3,4 Mammalian GDH utilizes either coenzyme with nearly identical ef®cacy. Bovine GDH is the most extensively studied ver- tebrate form and has been shown to exhibit com- plex homotropic and heterotropic allosteric regulation. Kinetic and binding analyses have clearly shown that NAD  and NADP  exhibit negatively cooperative binding in the presence of glutamate or glutarate. 5±7 In the reductive amin- ation reaction, evidence for such negative coopera- tivity is only found in binding studies. 6,8 NAD(P)H binding is clearly negatively cooperative at pH 8.0, but not at pH 7.0. In the presence of glutamate, NAD(P)H binding is greatly enhanced and exhibits negative cooperativity at both pHs. In the presence E-mail address of the corresponding author: tom@bragg.bio.purdue.edu doi:10.1006/jmbi.2001.4499 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 707±720 0022-2836/01/0020707±14 $35.00/0 # 2001 Academic Press