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Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase.

ABSTRACT: Glutamate dehydrogenase (GDH) is a target for treating insulin-related disorders, such as hyperinsulinism hyperammonemia syndrome. Modeling native ligand binding has shown promise in designing GDH inhibitors and activators. Our computational investigation of the nicotinamide adenine diphosphate hydride (NADH)/adenosine diphosphate (ADP) site presented in this paper provides insight into the opposite allosteric effects induced at a single site of binding inhibitor NADH versus activator ADP to GDH. The computed binding free-energy difference between NADH and ADP using thermodynamic integration is -0.3 kcal/mol, which is within the -0.275 and?-1.7 kcal/mol experimental binding free-energy difference range. Our simulations show an interesting model of ADP with dissimilar binding conformations at each NADH/ADP site in the GDH trimer, which explains the poorly understood strong binding but weak activation shown in experimental studies. In contrast, NADH showed similar inhibitory binding conformations at each NADH/ADP site. The structural analysis of the important residues in the NADH/ADP binding site presented in this paper may provide potential targets for mutation studies for allosteric drug design.

SUBMITTER: Nassar OM

PROVIDER: S-EPMC6863735 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase.

Nassar Omneya M OM Wong Ka-Yiu KY Lynch Gillian C GC Smith Thomas J TJ Pettitt B Montgomery BM

Protein science : a publication of the Protein Society 20191101 12

Glutamate dehydrogenase (GDH) is a target for treating insulin-related disorders, such as hyperinsulinism hyperammonemia syndrome. Modeling native ligand binding has shown promise in designing GDH inhibitors and activators. Our computational investigation of the nicotinamide adenine diphosphate hydride (NADH)/adenosine diphosphate (ADP) site presented in this paper provides insight into the opposite allosteric effects induced at a single site of binding inhibitor NADH versus activator ADP to GDH ...[more]

PMID: 31610054

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Project description:Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia (HI/HA) syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of allosteric network behind GDH regulation, identifications of allosteric factors and associated mechanisms are paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our structural analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of the inhibitor GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bound GDH activates a triangular allosteric network, interlinking the inhibitor with regulatory and catalytic sites. This allostery produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. In silico mutagenesis examinations further underpin the molecular basis of HI/HA dominant mutations and consequent over-activity of GDH through alteration of this allosteric communication network. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

Project description:Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.

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Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase.

Publications

Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase.

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