Filament formation drives catalysis by glutaminase enzymes important in cancer progression.

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an 'activation loop' to assume a specific conformation that works together with a 'lid' to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

Clinical, Biomarker, and Research Tests Among US Government Personnel and Their Family Members Involved in Anomalous Health Incidents.

Importance: Since 2015, US government and related personnel have reported dizziness, pain, visual problems, and cognitive dysfunction after experiencing intrusive sounds and head pressure. The US government has labeled these anomalous health incidents (AHIs). Objective: To assess whether participants with AHIs differ significantly from US government control participants with respect to clinical, research, and biomarker assessments. Design, Setting, and Participants: Exploratory study conducted between June 2018 and July 2022 at the National Institutes of Health Clinical Center, involving 86 US government staff and family members with AHIs from Cuba, Austria, China, and other locations as well as 30 US government control participants. Exposures: AHIs. Main Outcomes and Measures: Participants were assessed with extensive clinical, auditory, vestibular, balance, visual, neuropsychological, and blood biomarkers (glial fibrillary acidic protein and neurofilament light) testing. The patients were analyzed based on the risk characteristics of the AHI identifying concerning cases as well as geographic location. Results: Eighty-six participants with AHIs (42 women and 44 men; mean [SD] age, 42.1 [9.1] years) and 30 vocationally matched government control participants (11 women and 19 men; mean [SD] age, 43.8 [10.1] years) were included in the analyses. Participants with AHIs were evaluated a median of 76 days (IQR, 30-537) from the most recent incident. In general, there were no significant differences between participants with AHIs and control participants in most tests of auditory, vestibular, cognitive, or visual function as well as levels of the blood biomarkers. Participants with AHIs had significantly increased fatigue, depression, posttraumatic stress, imbalance, and neurobehavioral symptoms compared with the control participants. There were no differences in these findings based on the risk characteristics of the incident or geographic location of the AHIs. Twenty-four patients (28%) with AHI presented with functional neurological disorders. Conclusions and Relevance: In this exploratory study, there were no significant differences between individuals reporting AHIs and matched control participants with respect to most clinical, research, and biomarker measures, except for objective and self-reported measures of imbalance and symptoms of fatigue, posttraumatic stress, and depression. This study did not replicate the findings of previous studies, although differences in the populations included and the timing of assessments limit direct comparisons.

Neuroimaging Findings in US Government Personnel and Their Family Members Involved in Anomalous Health Incidents.

Importance: US government personnel stationed internationally have reported anomalous health incidents (AHIs), with some individuals experiencing persistent debilitating symptoms. Objective: To assess the potential presence of magnetic resonance imaging (MRI)-detectable brain lesions in participants with AHIs, with respect to a well-matched control group. Design, Setting, and Participants: This exploratory study was conducted at the National Institutes of Health (NIH) Clinical Center and the NIH MRI Research Facility between June 2018 and November 2022. Eighty-one participants with AHIs and 48 age- and sex-matched control participants, 29 of whom had similar employment as the AHI group, were assessed with clinical, volumetric, and functional MRI. A high-quality diffusion MRI scan and a second volumetric scan were also acquired during a different session. The structural MRI acquisition protocol was optimized to achieve high reproducibility. Forty-nine participants with AHIs had at least 1 additional imaging session approximately 6 to 12 months from the first visit. Exposure: AHIs. Main Outcomes and Measures: Group-level quantitative metrics obtained from multiple modalities: (1) volumetric measurement, voxel-wise and region of interest (ROI)-wise; (2) diffusion MRI-derived metrics, voxel-wise and ROI-wise; and (3) ROI-wise within-network resting-state functional connectivity using functional MRI. Exploratory data analyses used both standard, nonparametric tests and bayesian multilevel modeling. Results: Among the 81 participants with AHIs, the mean (SD) age was 42 (9) years and 49% were female; among the 48 control participants, the mean (SD) age was 43 (11) years and 42% were female. Imaging scans were performed as early as 14 days after experiencing AHIs with a median delay period of 80 (IQR, 36-544) days. After adjustment for multiple comparisons, no significant differences between participants with AHIs and control participants were found for any MRI modality. At an unadjusted threshold (P < .05), compared with control participants, participants with AHIs had lower intranetwork connectivity in the salience networks, a larger corpus callosum, and diffusion MRI differences in the corpus callosum, superior longitudinal fasciculus, cingulum, inferior cerebellar peduncle, and amygdala. The structural MRI measurements were highly reproducible (median coefficient of variation <1% across all global volumetric ROIs and <1.5% for all white matter ROIs for diffusion metrics). Even individuals with large differences from control participants exhibited stable longitudinal results (typically, <±1% across visits), suggesting the absence of evolving lesions. The relationships between the imaging and clinical variables were weak (median Spearman ρ = 0.10). The study did not replicate the results of a previously published investigation of AHIs. Conclusions and Relevance: In this exploratory neuroimaging study, there were no significant differences in imaging measures of brain structure or function between individuals reporting AHIs and matched control participants after adjustment for multiple comparisons.

Alone and together: current approaches to targeting glutaminase enzymes as part of anti-cancer therapies.

Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.

Many strategies exist to inhibit cancer progression, from chemotherapy to more targeted therapies that exploit differences between tumors and healthy tissue. One such targeted strategy involves inhibition of the enzyme glutaminase, which converts glutamine obtained from the bloodstream into nutrients that fuel tumor growth. Research into glutaminase is ongoing, with regulation of the enzyme, and novel molecular approaches to inhibit its activity, being key focus areas. Here, we review recent progress on targeting glutaminase enzymes for anti-cancer therapy, including several approaches in which glutaminase inhibitors are combined with inhibitors of other cancer-relevant targets, to increase the overall effectiveness of the treatment.

The risk of fragment penetrating injury to the heart.

Injury due to the penetration of fragments into parts of the body has been the major cause of morbidity and mortality after an explosion. Penetrating injuries into the heart present very high mortality, yet the risk associated with such injuries has not been quantified. Quantifying this risk is key in the design of personal protection and the design of infrastructure. This study is the first quantitative assessment of cardiac penetrating injuries from energised fragments. Typical fragments (5-mm sphere, 0.78-g right-circular cylinder and 1.1-g chisel-nosed cylinder) were accelerated to a range of target striking velocities using a bespoke gas-gun system and impacted ventricular and atrial walls of lamb hearts. The severity of injury was shown to not depend on location (ventricular or atrial wall). The striking velocity with 50% probability of critical injury (Abbreviated Injury Scale (AIS) 5 score) ranged between 31 and 36 m/s across all 3 fragments used. These findings can help directly in reducing morbidity and mortality from explosive events as they can be implemented readily into models that aim to predict casualties in an explosive event, inform protocols for first responders, and improve design of infrastructure and personal protective equipment.

Filament formation drives catalysis by glutaminase enzymes important in cancer progression.

The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the 'glutamine addiction' of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide an unprecedented view of the conformational states essential for catalysis. Filament formation guides an 'activation loop' to assume a specific conformation that works together with a 'lid' to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.

Mediation Effects of Premarital Sexual Permissiveness on the Relationship Between Expectations for Marriage and Marital Intention of Vietnamese Undergraduate Students.

Purpose: The purpose of this study was to investigate the direct and indirect effects of expectations for marital relationships and premarital sexual permissiveness on intent to marry of Vietnamese emerging adults. Patients and Methods: Our cross-sectional study was focused on emerging adults including 344 participants, undergraduate students from universities in Viet Nam. This study was assessed by using the PLS-SEM approach. Results: The main findings demonstrated that (i) sexual orientation have a significant effect on marital intention; (ii) individuals' expectations for marital relationship have a direct effect on marital intention; and (iii) premarital sexuality permissiveness mediates the relationship between expectations for marital relationship and marital intention. Conclusion: Our results contribute important documents and clearer understanding of emerging adults' expectations and requirements in a relationship for the marriage decision-making process.

Non-Lethal Blasts can Generate Cavitation in Cerebrospinal Fluid While Severe Helmeted Impacts Cannot: A Novel Mechanism for Blast Brain Injury.

Cerebrospinal fluid (CSF) cavitation is a likely physical mechanism for producing traumatic brain injury (TBI) under mechanical loading. In this study, we investigated CSF cavitation under blasts and helmeted impacts which represented loadings in battlefield and road traffic/sports collisions. We first predicted the human head response under the blasts and impacts using computational modelling and found that the blasts can produce much lower negative pressure at the contrecoup CSF region than the impacts. Further analysis showed that the pressure waves transmitting through the skull and soft tissue are responsible for producing the negative pressure at the contrecoup region. Based on this mechanism, we hypothesised that blast, and not impact, can produce CSF cavitation. To test this hypothesis, we developed a one-dimensional simplified surrogate model of the head and exposed it to both blasts and impacts. The test results confirmed the hypothesis and computational modelling of the tests validated the proposed mechanism. These findings have important implications for prevention and diagnosis of blast TBI.

High-resolution structures of mitochondrial glutaminase C tetramers indicate conformational changes upon phosphate binding.

The mitochondrial enzyme glutaminase C (GAC) is upregulated in many cancer cells to catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. The dependence of cancer cells on this transformed metabolic pathway highlights GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators such as inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescent reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the Y466W GAC tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric Y466W GAC bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e., the "lid"), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates, resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the Y466W GAC tetramer exhibited bound glutamine with closed lids. Glutamine can bind with high affinity to each subunit, which subsequently undergo simultaneous catalysis. These findings explain how the regulated transitioning of GAC between different conformational states ensures that maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.

Penetration of Energized Metal Fragments to Porcine Thoracic Tissues.

Energized fragments from explosive devices have been the most common mechanism of injury to both military personnel and civilians in recent conflicts and terrorist attacks. Fragments that penetrate into the thoracic cavity are strongly associated with death due to the inherent vulnerability of the underlying structures. The aim of this study was to investigate the impact of fragment-simulating projectiles (FSPs) to tissues of the thorax in order to identify the thresholds of impact velocity for perforation through these tissues and the resultant residual velocity of the FSPs. A gas-gun system was used to launch 0.78-g cylindrical and 1.13-g spherical FSPs at intact porcine thoracic tissues from different impact locations. The sternum and rib bones were the most resistant to perforation, followed by the scapula and intercostal muscle. For both FSPs, residual velocity following perforation was linearly proportional to impact velocity. These findings can be used in the development of numerical tools for predicting the medical outcome of explosive events, which in turn can inform the design of public infrastructure, of personal protection, and of medical emergency response.

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

The Injury Mechanism of Traumatic Amputation.

Traumatic amputation has been one of the most defining injuries associated with explosive devices. An understanding of the mechanism of injury is essential in order to reduce its incidence and devastating consequences to the individual and their support network. In this study, traumatic amputation is reproduced using high-velocity environmental debris in an animal cadaveric model. The study findings are combined with previous work to describe fully the mechanism of injury as follows. The shock wave impacts with the casualty, followed by energised projectiles (environmental debris or fragmentation) carried by the blast. These cause skin and soft tissue injury, followed by skeletal trauma which compounds to produce segmental and multifragmental fractures. A critical injury point is reached, whereby the underlying integrity of both skeletal and soft tissues of the limb has been compromised. The blast wind that follows these energised projectiles completes the amputation at the level of the disruption, and traumatic amputation occurs. These findings produce a shift in the understanding of traumatic amputation due to blast from a mechanism predominately thought mediated by primary and tertiary blast, to now include secondary blast mechanisms, and inform change for mitigative strategies.

Characterizing upper limb function in the context of activities of daily living in CLN3 disease.

In CLN3 disease, impairments in motor function are frequently reported to have later onset compared to visual and cognitive decline, but upper limb motor function has yet to be explored in this population. In a cohort of 22 individuals with CLN3, we used a novel application of multiple measures to (1) characterize motor function, particularly of the upper limbs, in activities of daily living (ADLs), and (2) explore associations between motor function and age as well as visual ability, disease severity, and cognitive function, as evaluated by the Unified Batten Disease Rating Scale (UBDRS), a validated CLN3 disease measure. ADLs that required coordination, speed, and fine motor control were particularly challenging for children with CLN3 based on item-level performance across direct assessments (Jebsen-Taylor Hand Function Test [JTHFT] and MyoSet Tools) and caregiver reports (Pediatric Evaluation of Disability Inventory-Computer Adaptive Testing [PEDI-CAT] and Patient-Reported Outcomes Measurement Information System [PROMIS] Pediatric Upper Extremity). Poorer visual ability, disease severity, and cognitive function were associated with worse performance on these measures, whereas age had limited impact. These findings support the need for children with CLN3 to receive skilled clinical evaluation and treatment tailored to their individual needs, particularly in the context of ADLs, as their symptom profile progresses.

Pelvic Protection Limiting Lower Limb Flail Reduces Mortality.

Pelvic blast injury is one of the most severe patterns of injury to be sustained by casualties of explosions. We have previously identified the mechanism of injury in a shock tube-mediated murine model, linking outward flail of the lower limbs to unstable pelvic fractures and vascular injury. As current military pelvic protection does not protect against lower limb flail, in this study we have utilized the same murine model to investigate the potential of novel pelvic protection to reduce injury severity. Fifty cadaveric mice underwent shock-tube blast testing and subsequent injury analysis. Pelvic protection limiting lower limb flail resulted in a reduction of pelvic fracture incidence from both front-on (relative risk (RR) 0.5, 95% confidence intervals (CIs) 0.3-0.9, p < 0.01) and under-body (RR 0.3, 95% CI 0.1-0.8 p < 0.01) blast, with elimination of vascular injury in both groups (p < 0.001). In contrast, pelvic protection, which did not limit flail, had no effect on fracture incidence compared to the control group and was only associated with a minimal reduction in vascular injury (RR 0.6, 95% CI 0.4-1.0, p < 0.05). This study has utilized a novel strategy to provide proof of concept for the use of pelvic protection, which limits limb flail to mitigate the effects of pelvic blast injury.

Mapping the Risk of Fracture of the Tibia From Penetrating Fragments.

Penetrating injuries are commonly inflicted in attacks with explosive devices. The extremities, and especially the leg, are the most commonly affected body areas, presenting high risk of infection, slow recovery, and threat of amputation. The aim of this study was to quantify the risk of fracture to the anteromedial, posterior, and lateral aspects of the tibia from a metal fragment-simulating projectile (FSP). A gas gun system and a 0.78-g cylindrical FSP were employed to perform tests on an ovine tibia model. The results from the animal study were subsequently scaled to obtain fracture-risk curves for the human tibia using the cortical thickness ratio. The thickness of the surrounding soft tissue was also taken into account when assessing fracture risk. The lateral cortex of the tibia was found to be most susceptible to fracture, whose impact velocity at 50% risk of EF1+, EF2+, EF3+, and EF4+ fracture types - according to the modified Winquist-Hansen classification - were 174, 190, 212, and 282 m/s, respectively. The findings of this study will be used to increase the fidelity of predictive models of projectile penetration.

A New Understanding of the Mechanism of Injury to the Pelvis and Lower Limbs in Blast.

Dismounted complex blast injury (DCBI) has been one of the most severe forms of trauma sustained in recent conflicts. This injury has been partially attributed to limb flail; however, the full causative mechanism has not yet been fully determined. Soil ejecta has been hypothesized as a significant contributor to the injury but remains untested. In this study, a small-animal model of gas-gun mediated high velocity sand blast was used to investigate this mechanism. The results demonstrated a correlation between increasing sand blast velocity and injury patterns of worsening severity across the trauma range. This study is the first to replicate high velocity sand blast and the first model to reproduce the pattern of injury seen in DCBI. These findings are consistent with clinical and battlefield data. They represent a significant change in the understanding of blast injury, producing a new mechanistic theory of traumatic amputation. This mechanism of traumatic amputation is shown to be high velocity sand blast causing the initial tissue disruption, with the following blast wind and resultant limb flail completing the amputation. These findings implicate high velocity sand blast, in addition to limb flail, as a critical mechanism of injury in the dismounted blast casualty.

Gelatine Backing Affects the Performance of Single-Layer Ballistic-Resistant Materials Against Blast Fragments.

Penetrating trauma by energized fragments is the most common injury from explosive devices, the main threat in the contemporary battlefield. Such devices produce projectiles dependent upon their design, including preformed fragments, casings, glass, or stones; these are subsequently energized to high velocities and cause serious injuries to the body. Current body armor focuses on the essential coverage, which is mainly the thoracic and abdominal area, and can be heavy and cumbersome. In addition, there may be coverage gaps that can benefit from the additional protection provided by one or more layers of lightweight ballistic fabrics. This study assessed the performance of single layers of commercially available ballistic protective fabrics such as Kevlar®, Twaron®, and Dyneema®, in both woven and knitted configurations. Experiments were carried out using a custom-built gas-gun system, with a 0.78-g cylindrical steel fragment simulating projectile (FSP) as the impactor, and ballistic gelatine as the backing material. FSP velocity at 50% risk of material perforation, gelatine penetration, and high-risk wounding to soft tissue, as well as the depth of penetration (DoP) against impact velocity and the normalized energy absorption were used as metrics to rank the performance of the materials tested. Additional tests were performed to investigate the effect of not including a soft-tissue simulant backing material on the performance of the fabrics. The results show that a thin layer of ballistic material may offer meaningful protection against the penetration of this FSP. Additionally, it is essential to ensure a biofidelic boundary condition as the protective efficacy of fabrics was markedly altered by a gelatine backing.

The risk of fracture to the tibia from a fragment simulating projectile.

Penetrating injuries due to fragments energised by an explosive event are life threatening and are associated with poor clinical and functional outcomes. The tibia is the long bone most affected in survivors of explosive events, yet the risk of penetrating injury to it has not been quantified. In this study, an injury-risk assessment of penetrating injury to the tibia was conducted using a gas-gun system with a 0.78-g cylindrical fragment simulating projectile. An ovine tibia model was used to generate the injury-risk curves and human cadaveric tests were conducted to validate and scale the results of the ovine model. The impact velocity at 50% risk (±95% confidence intervals) for EF1+, EF2+, EF3+, and EF4+ fractures to the human tibia - using the modified Winquist-Hansen classification - was 271 ± 30, 363 ± 46, 459 ± 102, and 936 ± 182 m/s, respectively. The scaling factor for the impact velocity from cadaveric ovine to human was 2.5. These findings define the protection thresholds to improve the injury outcomes for fragment penetrating injury to the tibia.

The activation loop and substrate-binding cleft of glutaminase C are allosterically coupled.

The glutaminase C (GAC) isoform of mitochondrial glutaminase is overexpressed in many cancer cells and therefore represents a potential therapeutic target. Understanding the regulation of GAC activity has been guided by the development of spectroscopic approaches that measure glutaminase activity in real time. Previously, we engineered a GAC protein (GAC(F327W)) in which a tryptophan residue is substituted for phenylalanine in an activation loop to explore the role of this loop in enzyme activity. We showed that the fluorescence emission of Trp-327 is enhanced in response to activator binding, but quenched by inhibitors of the BPTES class that bind to the GAC tetramer and contact the activation loop, thereby constraining it in an inactive conformation. In the present work, we took advantage of a tryptophan substitution at position 471, proximal to the GAC catalytic site, to examine the conformational coupling between the activation loop and the substrate-binding cleft, separated by ∼16 Å. Comparison of glutamine binding in the presence or absence of the BPTES analog CB-839 revealed a reciprocal relationship between the constraints imposed on the activation loop position and the affinity of GAC for substrate. Binding of the inhibitor weakened the affinity of GAC for glutamine, whereas activating anions such as Pi increased this affinity. These results indicate that the conformations of the activation loop and the substrate-binding cleft in GAC are allosterically coupled and that this coupling determines substrate affinity and enzymatic activity and explains the activities of CB-839, which is currently in clinical trials.