Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

Molecular dynamics calculations and bionformatic studies of dihydrofolate reductase (DHFR) have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. Experimental studies demonstrated that distal residues G121, M42, and F125 in E. coli DHFR participate in that network. The missing link in our understanding of DHFR catalysis is the lack of a mechanism by which such remote residues can affect the catalyzed chemistry at the active site. Here, we present a study of the temperature dependence of intrinsic kinetic isotope effects (KIEs) that indicates synergism between a remote residue in that dynamic network, G121, and the active site’s residue I14. The intrinsic KIEs for the I14A–G121V double mutant showed steeper temperature dependence (Δ<i>E</i>_a(T‑H)) than expected from comparison of the wild type and two single mutants. That effect was nonadditive (i.e., Δ<i>E</i>_{a(T‑H) G121V} + Δ<i>E</i>_{a(T‑H) I14A} < Δ<i>E</i>_{a(T‑H) double mutant}), which indicates a synergism between the two residues. This finding links the remote residues in the network under investigation to the enzyme’s active site, providing a mechanism by which these residues can be coupled to the catalyzed chemistry. This experimental evidence validates calculations proposing that both remote and active site residues constitute a network of coupled promoting motions correlated to the bond activation step (C–H → C hydride transfer in this case). Additionally, the effect of I14A and G121V mutations on single turnover rates was additive rather than synergistic. Although single turnover rate measurements are more readily available and thus more popular than assessing intrinsic KIEs, the current finding demonstrates that these rates, which in DHFR reflect several microscopic rate constants, can fall short of revealing the nature of the C–H bond activation per se

Extension and Limits of the Network of Coupled Motions Correlated to Hydride Transfer in Dihydrofolate Reductase

Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a “global dynamic network” of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts

Structural and Kinetic Studies of Formate Dehydrogenase from <i>Candida boidinii</i>

The structure of formate dehydrogenase from <i>Candida boidinii</i> (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO₂ fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD⁺) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10^–7 (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented

Burden of illness for super-refractory status epilepticus patients

<p><b>Objective:</b> To provide an estimate of the annual number of super-refractory status epilepticus (SRSE) cases in the United States (US) and to evaluate utilization of hospital resources by these patients.</p> <p><b>Methods:</b> The Premier Hospital Database was utilized to estimate the number of SRSE cases based on hospital discharges during 2012. Discharges were classified as SRSE cases based on an algorithm using seizure-related International Classification of Diseases-9 (ICD-9) codes, Intensive Care Unit (ICU) length of stay (LOS), and treatment protocols (e.g., benzodiazepines, antiepileptic drugs (AEDs), and ventilator use). Secondary analyses were conducted using more restrictive algorithms for SRSE.</p> <p><b>Results:</b> A total of 6,325 hospital discharges were classified as SRSE cases from a total of 5,300,000 hospital discharges. Applying a weighting based on hospital characteristics and 2012 US demographics, this projected to an estimated 41,156 cases of SRSE in the US during 2012, an estimated incidence rate of approximately 13/100,000 annually for SRSE in the US. Secondary analyses using stricter SRSE algorithms resulted in estimated incidence rates of approximately 11/100,000 and 8/100,000 annually. The mean LOS for SRSE hospitalizations was 16.5 days (median = 11; interquartile range [IQR] = 6–20), and the mean LOS in ICU was 9.3 days (median = 6; IQR = 3–12). The mean cost of an SRSE hospitalization was $51,247 (median=$33,294; 95% CI=$49,634–$52,861).</p> <p><b>Limitations:</b> The analysis uses ICD-9 diagnostic codes and claims information, and there are inherent limitations in any methodology based on treatment protocol, which created challenges in distinguishing with complete accuracy between SRSE, RSE, and SE on the basis of care patterns in the database.</p> <p><b>Conclusion:</b> SRSE is associated with high mortality and morbidity, which place a high burden on healthcare resources. Projections based upon the findings of this study suggest an estimated 25,821 to 41,959 cases of SRSE may occur in the US each year, but more in-depth studies are required.</p

Current and Future Disease Progression of the Chronic HCV Population in the United States

<div><p>Chronic hepatitis C virus (HCV) infection can lead to advanced liver disease (AdvLD), including cirrhosis, decompensated cirrhosis, and liver cancer. The aim of this study was to determine recent historical rates of HCV patient progression to AdvLD and to project AdvLD prevalence through 2015. We first determined total 2008 US chronic HCV prevalence from the National Health and Nutrition Evaluation Surveys. Next, we examined disease progression and associated non-pharmacological costs of diagnosed chronic HCV-infected patients between 2007–2009 in the IMS LifeLink and CMS Medicare claims databases. A projection model was developed to estimate AdvLD population growth through 2015 in patients diagnosed and undiagnosed as of 2008, using the 2007–2009 progression rates to generate a “worst case” projection of the HCV-related AdvLD population (i.e., scenario where HCV treatment is the same in the forecasted period as it was before 2009). We found that the total diagnosed chronic HCV population grew from 983,000 to 1.19 million in 2007–2009, with patients born from 1945–1964 accounting for 75.0% of all patients, 83.7% of AdvLD patients, and 79.2% of costs in 2009, indicating that HCV is primarily a disease of the “baby boomer” population. Non-pharmacological costs grew from $7.22 billion to$8.63 billion, with the majority of growth derived from the 60,000 new patients that developed AdvLD in 2007–2009, 91.5% of whom were born between 1945 and 1964. The projection model estimated the total AdvLD population would grow from 195,000 in 2008 to 601,000 in 2015, with 73.5% of new AdvLD cases from patients undiagnosed as of 2008. AdvLD prevalence in patients diagnosed as of 2008 was projected to grow 6.5% annually to 303,000 patients in 2015. These findings suggest that strategies to diagnose and treat HCV-infected patients are urgently needed to increase the likelihood that progression is interrupted, particularly for patients born from 1945–1964.</p></div