Allosteric regulation of glutamate dehydrogenase deamination activity

Abstract

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.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

“…The binding site of palmitoyl-CoA on GDH is still unknown, making it difficult to infer structural underpinnings of these results. However, the significant increase in rescue if mitoNEET is bound before either coenzyme binds may indirectly indicate that mitoNEET stabilizes the open conformation of GDH and/or that palmitoyl-CoA hastens the loss of the [2Fe-2S] cluster from mitoNEET and allows the now available ligating cysteine residues to modify GDH as a mechanism of rescue [ 13 ].…”

“…GTP is the best understood inhibitor of GDH [ 4 , 13 , 20 ]. Mutation(s) in the GDH enzyme that negate the negative allostery of GTP and increase the activating allostery of leucine are known to cause the clinical condition of hyperinsulinemia/hyperammonemia (HI/HA).…”

“…HI/HA is characterized by hypoglycemia and plasma ammonia levels that are 3–5 times higher than physiologically normal [ 20 ]. Due to the physiological relevance of understanding the inhibition of GDH by GTP to human health and disease, it is not surprising that a substantial amount of work has been done to understand the biochemistry and biophysics of this phenomenon [ 2 , 13 ]. GTP alone does not bind tightly to GDH, but works synergistically with NAD in the regulatory site to shift GDH to the closed, or abortive, conformation [ 13 ].…”

Structural and Functional Analysis of Proteins Using Rigidity Theory

“…We now briefly discuss and review an important application of rigidity theory for analysis of allosteric signalling in protein structures. Allostery is one of the most powerful and fundamental mechanisms regulating protein function [8][9][10][11][12][42][43][44]. Allostery refers to the regulation of protein function at a distance, where a perturbation of a protein structure at one part of protein structure (for example, due to a binding or mutational event) can affect conformations and dynamics at another distant site, resulting in regulation of protein function.…”

“…One of the important areas in allostery research is describing the physical mechanism of distant coupled conformational changes. The utilization and extension of our earlier fundamental work in modelling allostery in frameworks and graphs [16] and a first rigidity-based mechanistic model of allosteric signalling has led to several important breakthroughs in understanding how allostery controls enzyme and receptor function [11,12,24,44]. Our rigidity theory methods predict that if mechanical perturbation of rigidity at one site of the protein can transmit and propagate across a protein structure and, in turn, cause a change in the available conformational degrees of freedom and a change in the conformation and dynamics at a second distant site, resulting in allosteric transmission (Fig.…”