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Aims: To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel‐derived glycerol (Gly) into added‐value compounds. Methods and Results: Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single‐cell oil (SCO), endopolysaccharides and polyols when these micro‐organisms were grown on biodiesel‐derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB‐252 and Cryptococcus curvatus NRRL Y‐1511. At initial Gly (Gly0) ≈40 g l−1, most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non‐negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB‐252 produced 7·2 g l−1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch‐bioreactor experiment with Gly0 ≈80 g l−1, produced 30–33 g l−1 of DCW and ~30 g l−1 of Ara. At shake‐flasks with Gly0 ≈125 g l−1, Ara of ~48 g l−1 (conversion yield of polyol on Gly consumed ≈0·62 g g−1) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. Conclusions: Newly isolated, food‐derived and non‐previously studied yeast isolates converted biodiesel‐derived Gly into several added‐value metabolites. Significance and Impact of the Study: Alternative ways of crude Gly valorization through yeast fermentations were provided and added‐value compounds were synthesized. [ABSTRACT FROM AUTHOR]

Our purpose was to determine the efficacy of irinotecan plus paclitaxel administered on day 1, repeated every 2 weeks, in untreated patients with advanced or metastatic non-small-cell lung cancer (NSCLC). In total, 56 patients with inoperable or metastatic stage III and IV NSCLC with a histologically or cytologically confirmed diagnosis were enrolled. None of the patients had undergone prior chemotherapy or radiation therapy. Treatment involved irinotecan 125 mg m(-2) and paclitaxel 135 mg m(-2) administered on day 1 and repeated every 2 weeks for a planned number of nine cycles. With a standard dose of paclitaxel at 135 mg m(-2), the dosage of irinotecan was escalated at four levels: 75, 100, 125 and 150 mg m(-2); 125 mg m(-2) was established as the maximum tolerated dose; this dosage was administered to 46 patients. A total of 52 patients (median age 65 years, range 38-77 years) were assessable for toxicity and survival and 46 for response rate. Out of 46 evaluable patients, 19 achieved partial response (41.3%), 17 had stable disease (37%) and 10 (21.7%) experienced disease progression. The median duration of response was 6 months (range 2-9+ months). The main adverse reactions were myelotoxicity (grades 3 and 4) in 10 (19.2%) patients and diarrhoea (grade 3) in four (7.7%) patients. Irinotecan combined with paclitaxel, administered every 2 weeks, appears to be an effective treatment for advanced-stage NSCLC. [ABSTRACT FROM AUTHOR]

Substance P release by enteric nerves could be an initiating factor for mucosal mast cell (MMC) activation that is associated with weaning in the rat. Capsaicin, which depletes substance P from enteric nerves, should therefore prevent MMC degranulation. Rat pups received either capsaicin (50 mg/kg) or vehicle control subcutaneous injections at 3 and 6 days of life. Capsaicin-treated and control litters were killed at 16, 18, 20, 22, 24 and 26 days of life. MMC activation was measured by serum levels of rat mast cell protease II (RMCPII). Intestinal development was assessed by microdissection to measure villus area, crypt length and crypt cell production rate. RMCPII levels were similar in capsaicin-treated and control rats and peaked at day 22 of life, and intestinal development was not retarded by capsaicin treatment. We conclude that substance P release is unlikely to be an initiating factor for the MMC activation that is associated with weaning. [ABSTRACT FROM AUTHOR]

To determine the effects of intravenous nitroglycerin on the velocities and excursions of the acutely ischemic myocardium, 20 open-chest dogs were studied by use of ultrasound. In 10 dogs with acute septal ischemia, the posterior wall excursion during contraction (B-C excursion), the mean systolic posterior wall velocity, and the posterior wall excursion remained unaltered. Nitroglycerin, however, increased all these parameters (P c 0.01). In 10 dogs with acute posterior wall ischemia the B-C excursion (aneurysmal bulging) increased (P < 0.01), but the mean systolic posterior wall velocity and posterior wall excursion decreased (P < 0.01). Nitroglycerin increased even more the aneurysmal bulging (P < 0.01) and the other parameters (P < 0.01). Increased regional blood flow, reduced afterload, and mechanical pulling of the ischemic myocardium seem to be a possible mechanism. The measurements were obtained using the recently described method of the specific points. [ABSTRACT FROM AUTHOR]

In 100 fertile men 2% were found to have sperm-agglutinating antibodies but these were not clearyly related in any particuliue case of their infertility. Six weeks after vasecomy 6% of the 100 fertile men hand magglunationg antibodies. The significance and importance of these findings are unknown. [ABSTRACT FROM AUTHOR]

Pancreatic cystic lesions are rare clinical entities. To the best of our knowledge, only 38 cases have been reported in the English literature in children under the age of 2 years. We present a 2-month-old infant with a cystic lesion in the head of pancreas. We reviewed the possible causes and present our dilemmas in the management of these patients. [ABSTRACT FROM AUTHOR]

Abstract Lung resection is the mainstay of treatment in patients with early stage non-small cell lung cancer. However, lung cancer patients often suffer from comorbidities and the respiratory reserve should be carefully evaluated preoperatively in order to avoid postoperative complications. Forced expiratory volume in 1 second (FEV1) is considered to be an index that depicts the patient's respiratory efficacy and its prediction has a key role in the preoperative evaluation of lung cancer patients with impaired lung function. Prediction of postoperative FEV1 is currently possible with the use of perfusion radionuclide lung scanning. Quantitative CT is the analysis of data acquired during normal chest CT scan using the system's software. By applying a dual threshold of -500 to -910 Hounsfield Units, functional lung volumes are estimated and postoperative FEV1 can be predicted by reducing the preoperative measurement by the fraction of the part to be resected. Studies have shown that preoperative predictions correlate well with the actual postoperative measurements. Additionally, quantitative CT results are in good agreement with perfusion scintigraphy predictions. Newer radiological techniques such as perfusion MRI and co-registered SPECT/CT have also been used in the preoperative evaluation with similar results. In conclusion, chest CT which is obligatory for staging, can be used for quantitative analysis of the already available data. It is technically simple, providing an accurate prediction of postoperative FEV1. Thus, quantitative CT appears to be a useful tool in the preoperative evaluation of lung cancer patients undergoing lung resection.

Abstract Introduction Hydrocarbon pneumonitis is an acute, intense pneumonitis resulting from aspiration of volatile hydrocarbon compounds with low viscosity and surface tension, most of which are members of the paraffin, naphthene and aromatic classes. Case presentation Six hours after participating in a party for teenagers, a 16-year-old boy developed dyspnea, cough, a fever (39°C) and chest pain. A chest radiograph showed infiltration in the right middle lobe. The patient reported alcohol abuse during the party and an episode of vomiting a few hours thereafter. He also reported practicing a fire-eating performance at the party using liquid paraffin, but was unaware of inhaling any of it. The radiographic infiltration was diagnosed as an aspiration pneumonia and he was treated at the local health center with antibiotics. Five days later, because of clinical deterioration, he was referred to a pulmonary clinic. A chest computed tomography scan was performed which showed consolidation with an air bronchogram in the right middle lobe and areas of atelectasis and ground glass opacities in the middle and lower right lobes. Spirometry revealed severe restriction of lung function. A bronchoscopy revealed inflamed, hyperemic mucosa. Bronchoalveolar lavage fluid revealed lipid-laden alveolar macrophages, which were detected by lipid staining, and neutrophilia. The patient was finally diagnosed with hydrocarbon pneumonitis and he was treated with systemic steroids and antibiotics. After 6 days of treatment there was complete clinical and significant radiologic regression. Conclusion Hydrocarbon pneumonitis should be included in the differential diagnosis of pneumonias. Recent exposure to volatile hydrocarbons provides a basis for clinical diagnosis, as symptoms and radiologic findings are not specific.

β-Lactamases are one of the primary enzymes responsible for antibiotic resistance and have existed for billions of years. The structural differences between a modern class A TEM-1 β-lactamase compared to a sequentially reconstructed Gram-negative bacteria β-lactamase are minor. Despite the similar structures and mechanisms, there are different functions between the two enzymes. We recently identified differences in dynamics effects that result from evolutionary changes that could potentially account for the increase in substrate specificity and catalytic rate. In this study, we used transition path sampling-based calculations of free energies to identify how evolutionary changes found between an ancestral β-lactamase, and its extant counterpart TEM-1 β-lactamase affect rate.

β -Lactamases are a class of well-studied enzymes that are known to have existed since billions of years ago, starting as a defense mechanism to stave off competitors and are now enzymes responsible for antibiotic resistance. Using ancestral sequence reconstruction, it is possible to study the crystal structure of a laboratory resurrected 2-3 billion year-old β -lactamase. Comparing the ancestral enzyme to its modern counterpart, a TEM-1 β -lactamase, the structural changes are minor, and it is probable that dynamic effects play an important role in the evolution of function. We used molecular dynamics simulations and employed transition path sampling methods to identify the presence of rate-enhancing dynamics at the femtosecond level in both systems, found that these fast motions are more efficiently coordinated in the modern enzyme, and examined how specific dynamics can pinpoint evolutionary effects that are essential for improving enzymatic catalysis., Competing Interests: The authors declare no competing financial interest.

Background: Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice., Methods: Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group)., Results: We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log 2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05)., Conclusions: Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here., (© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

The influence of protein motions on enzyme catalysis remains a topic of active discussion. Protein motions occur across a variety of time scales, from vibrational fluctuations in femtoseconds, to collective motions in milliseconds. There have been numerous studies that show conformational motions may assist in catalysis, protein folding, and substrate specificity. It is also known through transition path sampling studies that rapid promoting vibrations contribute to enzyme catalysis. Human purine nucleoside phosphorylase (PNP) is one enzyme that contains both an important conformational motion and a rapid promoting vibration. The slower motion in this enzyme is associated with a loop motion, that when open allows substrate entry and product release but closes over the active site during catalysis. We examine the differences between an unconstrained PNP structure and a PNP structure with constraints on the loop motion. To investigate possible coupling between the slow and fast protein dynamics, we employed transition path sampling, reaction coordinate identification, electric field calculations, and free energy calculations reported here.

Despite initial resistance, it has been increasingly accepted that protein dynamics plays a role in enzymatic catalysis. There have been two lines of research. Some works study slow conformational motions that are not coupled to the reaction coordinate, but guide the system towards catalytically competent conformations. Understanding at the atomistic level how this is accomplished has remained elusive except for a few systems. In this review we focus on fast sub-picosecond motions that are coupled to the reaction coordinate. The use of Transition Path Sampling has allowed us an atomistic description of how these rate-promoting vibrational motions are incorporated in the reaction mechanism. We will also show how we used insights from rate-promoting motions in protein design., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Simulation methods like transition path sampling (TPS) generate an abundance of information buried in the collection of reactive trajectories that they generate. However, only limited use has been made of this information, mainly for the identification of the reaction coordinate. The standard TPS tools have been designed for monitoring the progress of the system from reactants to products. However, the reaction coordinate does not contain all the information regarding the mechanism. In our earlier work, we have used TPS on enzymatic systems and have identified important motions in the reactant well that prepares the system for the reaction. Since these events take place in the reactant well, they are beyond the reach of standard TPS postprocessing methods. We present a simple scheme for identifying the common trends in enzymatic trajectories. This scheme was designed for a specific class of enzymatic reactions: it can be used for identifying motions that guide the system to reaction-ready conformations. We have applied it to two enzymatic systems that we have studied in the past, formate dehydrogenase and purine nucleoside phosphorylase, and we were able to identify interactions, far from the transition state, that are important for preparing the system for the reaction but that had been overlooked in earlier work.

Heavy enzyme isotope effects occur in proteins substituted with 2 H-, 13 C-, and 15 N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli ) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization., Competing Interests: The authors declare no competing interest.

We have analyzed the reaction catalyzed by formate dehydrogenase using transition path sampling. This system has recently received experimental attention using infrared spectroscopy and heavy-enzyme studies. Some of the experimental results point to the possible importance of protein motions that are coupled to the chemical step. We found that the residue Val123 that lies behind the nicotinamide ring occasionally comes into van der Waals contact with the acceptor and that in all reactive trajectories, the barrier-crossing event is preceded by this contact, meaning that the motion of Val123 is part of the reaction coordinate. Experimental results have been interpreted with a two-dimensional formula for the chemical rate, which cannot capture effects such as the one we describe.

A total of 11 yeast strains of Yarrowia lipolytica, Metschnikowia sp., Rhodotorula sp. and Rhodosporidium toruloides were grown under nitrogen-limited conditions with crude glycerol employed as substrate in shake flasks, presenting interesting dry cell weight (DCW) production. Three of these strains belonging to Metschnikowia sp. accumulated significant quantities of endopolysaccharides (i.e. the strain V.V.-D4 produced 11.0 g/L of endopolysaccharides, with polysaccharides in DCW ≈ 63% w/w). A total of six Y. lipolytica strains produced either citric acid or mannitol. Most of the screened yeasts presented somehow elevated lipid and polysaccharides in DCW values at the early steps of growth despite nitrogen appearance in the fermentation medium. Lipid in DCW values decreased as growth proceeded. R. toruloides DSM 4444 cultivated on media presenting higher glycerol concentrations presented interesting lipid-accumulating capacities (maximum lipid = 12.5 g/L, maximum lipid in DCW = 43.0-46.0% w/w, conversion yield on glycerol = 0.16 g/g). Replacement of crude glycerol by xylose resulted in somehow decreased lipid accumulation. In xylose/glycerol mixtures, xylose was more rapidly assimilated from glycerol. R. toruloides total lipids were mainly composed of triacylglycerols. Total cellular fatty acid composition on xylose presented some differences compared with that on glycerol. Cellular lipids contained mainly oleic and palmitic acid., (© FEMS 2020.)

The design of artificial enzymes is an emerging field of research. Although progress has been made, the catalytic proficiency of many designed enzymes is low compared to natural enzymes. Nevertheless, recently Hilvert et al. ( Nat. Chem. 2017, 9, 50-56) created a series of five artificial retro-aldolase enzymes via directed evolution, with the final variant exhibiting a rate comparable to the naturally occurring enzyme fructose 1,6 bisphosphate aldolase. We present a study of this system in atomistic detail that elucidates the effects of mutational changes on the chemical step. Transition path sampling is used to create ensembles of reactive trajectories, and committor analysis is used to identify the stochastic separatrix of each ensemble. The application of committor distribution analysis to constrained trajectories allows the identification of changes in important protein motions coupled to reaction across the generated series of the artificial retro-aldolases. We observed two different reaction mechanisms and analyzed the role of the residues participating in the reaction coordinate of each enzyme. However, only in the most evolved variant we identified a fast motion that promotes catalysis, suggesting that this rate promoting vibration was introduced during directed evolution. This study provides further evidence that protein dynamics must be taken into account in designing efficient artificial enzymes.

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13 C, 15 N, and nonexchangeable 2 H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly ( k chem light / k chem heavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster ( k chem light / k chem heavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster ( k chem light / k chem heavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts., Competing Interests: The authors declare no conflict of interest.

In recent years, there has been much discussion regarding the origin of enzymatic catalysis and whether including protein dynamics is necessary for understanding catalytic enhancement. An important contribution in this debate was made with the application of the vibrational Stark effect spectroscopy to measure electric fields in the active site. This provided a window on electric fields at the transition state in enzymatic reactions. We performed computational studies on two enzymes where we have shown that fast dynamics is part of the reaction mechanism and calculated the electric field near the bond-breaking event. We found that the fast motions that we had identified lead to an increase of the electric field, thus preparing an enzymatic configuration that is electrostatically favorable for the catalytic chemical step. We also studied the enzyme that has been the subject of Stark spectroscopy, ketosteroid isomerase, and found electric fields of a similar magnitude to the two previous examples.

In recent years, there has been encouraging progress in the engineering of enzymes that are designed to catalyze reactions not accelerated by natural enzymes. We tested the possibility of reengineering an existing enzyme by introducing a fast protein motion that couples to the reaction. Aromatic amine dehydrogenase is a system that has been shown to use a fast substrate motion as part of the reaction mechanism. We identified a mutation that preserves this fast motion but also introduces a favorable fast motion near the active site that did not exist in the native enzyme. Transition path sampling was used for the analysis of the atomic details of the mechanism.

Heavy-enzyme isotope effects ( 15 N-, 13 C-, and 2 H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect ( k chem light / k chem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect ( k chem light / k chem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage., Competing Interests: The authors declare no conflict of interest.

The emergence of minimally invasive surgery about 20 years ago revolutionized pediatric urology. Advances in pediatric devices allowed the widespread use of minimally invasive techniques in almost the entire range of pediatric urology. In this context, laparoscopy and later retroperitoneoscopy were developed and applied in a wide spectrum of urological diseases. Both approaches have since presented benefits and disadvantages that have been documented in various series. However, few comparative studies have been conducted. The aim of this review is to compare the two approaches and establish which is preferable in each field of pediatric urology., Competing Interests: The authors have no conflicts of interest to declare.

The relevance of sub-picosecond protein motions to the catalytic event remains a topic of debate. Heavy enzymes (isotopically substituted) provide an experimental tool for bond-vibrational links to enzyme catalysis. A recent transition path sampling study with heavy purine nucleoside phosphorylase (PNP) characterized the experimentally observed mass-dependent slowing of barrier crossing (Antoniou, D.; Ge, X.; Schramm, V. L.; Schwartz, S. D. J. Phys. Chem. Lett. 2012, 3, 3538). Here we computationally identify second-sphere amino acid residues predicted to influence the freedom of the catalytic site vibrational modes linked to heavy enzyme effects in PNP. We mutated heavy and light PNPs to increase the catalytic site vibrational freedom. Enzymatic barrier-crossing rates were converted from mass-dependent to mass-independent as a result of the mutations. The mutagenic uncoupling of femtosecond motions between catalytic site groups and reactants decreased transition state barrier crossing by 2 orders of magnitude, an indication of the femtosecond dynamic contributions to catalysis.

The definition of a transition state on an individual reactive trajectory is made via a committor analysis. In the past, the bottleneck definition has often been applied in configuration space. This is an approximation, and in order to expand this definition, we are revisiting an enzyme in which we had identified a fast subpicosecond motion that makes the reaction possible. First we used a time-series analysis method to identify the exact time when this motion initiates donor-acceptor compression. Then we modified the standard committor analysis of transition path sampling to identify events in phase space and found that there is a dividing surface in phase space significantly earlier than the configurationally defined transition-state crossing.

Escherichia coli dihydrofolate reductase (ecDHFR) is used to study fundamental principles of enzyme catalysis. It remains controversial whether fast protein motions are coupled to the hydride transfer catalyzed by ecDHFR. Previous studies with heavy ecDHFR proteins labeled with (13)C, (15)N, and nonexchangeable (2)H reported enzyme mass-dependent hydride transfer kinetics for ecDHFR. Here, we report refined experimental and computational studies to establish that hydride transfer is independent of protein mass. Instead, we found the rate constant for substrate dissociation to be faster for heavy DHFR. Previously reported kinetic differences between light and heavy DHFRs likely arise from kinetic steps other than the chemical step. This study confirms that fast (femtosecond to picosecond) protein motions in ecDHFR are not coupled to hydride transfer and provides an integrative computational and experimental approach to resolve fast dynamics coupled to chemical steps in enzyme catalysis.