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

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

Research output: Contribution to journalArticle

124 Citations (Scopus)

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

Fingerprint

Glutamate Dehydrogenase
NAD
Guanosine Triphosphate
Coenzymes
Adenosine Diphosphate
NADP
Phosphates
Allosteric Regulation
Hyperammonemia
purine
Deamination
Structural Models
Hyperinsulinism
Glutamic Acid
Catalytic Domain
Adenosine Triphosphate
Binding Sites

Keywords

  • Allostery
  • Glutamate dehydrogenase
  • Hyperinsulinism
  • Purine regulation

ASJC Scopus subject areas

  • Virology

Cite this

Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation. / Smith, Thomas; Peterson, Peter E.; Schmidt, Timothy; Fang, Jie; Stanley, Charles A.

In: Journal of Molecular Biology, Vol. 307, No. 2, 23.03.2001, p. 707-720.

Research output: Contribution to journalArticle

Smith, Thomas ; Peterson, Peter E. ; Schmidt, Timothy ; Fang, Jie ; Stanley, Charles A. / Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation. In: Journal of Molecular Biology. 2001 ; Vol. 307, No. 2. pp. 707-720.
@article{2c2ef2c62b1f4af2bb0787587414874a,
title = "Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation",
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.",
keywords = "Allostery, Glutamate dehydrogenase, Hyperinsulinism, Purine regulation",
author = "Thomas Smith and Peterson, {Peter E.} and Timothy Schmidt and Jie Fang and Stanley, {Charles A.}",
year = "2001",
month = "3",
day = "23",
doi = "10.1006/jmbi.2001.4499",
language = "English (US)",
volume = "307",
pages = "707--720",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation

AU - Smith, Thomas

AU - Peterson, Peter E.

AU - Schmidt, Timothy

AU - Fang, Jie

AU - Stanley, Charles A.

PY - 2001/3/23

Y1 - 2001/3/23

N2 - 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.

AB - 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.

KW - Allostery

KW - Glutamate dehydrogenase

KW - Hyperinsulinism

KW - Purine regulation

UR - http://www.scopus.com/inward/record.url?scp=0035937256&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035937256&partnerID=8YFLogxK

U2 - 10.1006/jmbi.2001.4499

DO - 10.1006/jmbi.2001.4499

M3 - Article

C2 - 11254391

AN - SCOPUS:0035937256

VL - 307

SP - 707

EP - 720

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -