Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation

Thomas J. Smith, Peter E. Peterson, Timothy Schmidt, Jie Fang, Charles A. Stanley

Research output: Contribution to journalArticle

127 Scopus citations

Abstract

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 efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety 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 refined and modified the previous structural model of allosteric regulation inferred from the original boGDH·NADH·GLU·GTP complex. The boGDH·NAD+·α-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 identified. 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.

Original languageEnglish (US)
Pages (from-to)707-720
Number of pages14
JournalJournal of Molecular Biology
Volume307
Issue number2
DOIs
StatePublished - Mar 23 2001
Externally publishedYes

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Keywords

  • Allostery
  • Glutamate dehydrogenase
  • Hyperinsulinism
  • Purine regulation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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