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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

A Genome-Scale Metabolic Model of Anabaena 33047 to Guide Genetic Modifications to Overproduce Nylon Monomers

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1985

Exercise hyperthermia as a factor limiting physical performance - Temperature effect on muscle metabolism

The effect of trunk cooling on the muscle contents of ATP, ADP, AMP, creatine phosphate (CrP), and creatine, as well as of glycogen, some glycolytic intermediates, pyruvate, and lactate were assessed in 11 fasted dogs exercised at 20 C on treadmill to exhaustion. Without cooling, dogs were able to run 57 min, and their rectal (Tre) and muscle (Tm) temperatures increased to 41.8 and 43.0 C, respectively. Cooling with ice packs prolonged the ability to run by 45 percent, and resulted in lower Tre (by 1.1 C) and Tm (by 1.2 C). Depletion of muscle content of total high-energy phosphates (ATP + CrP) and glycogen, and increases in contents of AMP, pyruvate, and lactate were lower in cooled dogs than in non-cooled dogs. The muscle content of lactiate correlated positively with TM. These results indicate that hypothermia accelerates glycolysis, and shifts the equilibrium between high- and low-energy phosphates in favor of the latter. The adverse effect of hypothermia on muscle metabolism may be relevant to the limitation of endurance.

Kozlowski, S.↗
NASA NTRS1992

Effects of microgravity and tail suspension on enzymes of individual soleus and tibialis anterior fibers

Selected enzymes of energy metabolism were measured in random individual fibers of soleus and tibialis anterior (TA) muscles from rats exposed for 2 wk to spaceflight (F) aboard Cosmos 2044 or tail suspension (T) and from synchronous controls. Average size of soleus fibers (dry weight per unit length) was reduced 37 percent in F and T fibers; there was little change in Ta fibers. Enzyme changes were more pronounced in soleus than in TA fibers. Three enzymes characteristic of fast-twitch muscles, pyruvate kinase, glycerol-3-phosphate dehydrogenase, and 1-phosphofructokinase, were elevated in F and T soleus fibers, but changes in phosphofructokinase were not statistically significant. In TA fibers analyzed for hexokinase, malate dehydrogenase, phosphohexoisomerase, and pyruvate kinase, only hexokinase and malate dehydrogenase showed significant changes. Hexokinase incresed 83 percent in one of two T muscles. Enzyme data for TA fibers typed by myosin adenosinetriphosphatase were more informative: phosphofructokinase, phosphorylase, and glycerol-3-phosphate dehydrogenase were increased in type IIn fibers of either F or T muscles or both. Malate dehydrogenase was not changed in fibers of any type in either F or T muscle.

Chi, Maggie M.-Y.↗
NASA NTRS1999

A Quantitative Study of Oxygen as a Metabolic Regulator

An acute reduction in oxygen (O2) delivery to a tissue is generally associated with a decrease in phosphocreatine, increases in ADP, NADH/NAD, and inorganic phosphate, increased rates of glycolysis and lactate production, and reduced rates of pyruvate and fatty acid oxidation. However, given the complexity of the human bioenergetic system and its components, it is difficult to determine quantitatively how cellular metabolic processes interact to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). Of special interest is the determination of mechanisms relating tissue oxygenation to observed metabolic responses at the tissue, organ, and whole body levels and the quantification of how changes in tissue O2 availability affect the pathways of ATP synthesis and the metabolites that control these pathways. In this study, we extend a previously developed mathematical model of human bioenergetics to provide a physicochemical framework that permits quantitative understanding of O2 as a metabolic regulator. Specifically, the enhancement permits studying the effects of variations in tissue oxygenation and in parameters controlling the rate of cellular respiration on glycolysis, lactate production, and pyruvate oxidation. The whole body is described as a bioenergetic system consisting of metabolically distinct tissue/organ subsystems that exchange materials with the blood. In order to study the dynamic response of each subsystem to stimuli, we solve the ordinary differential equations describing the temporal evolution of metabolite levels, given the initial concentrations. The solver used in the present study is the packaged code LSODE, as implemented in the NASA Lewis kinetics and sensitivity analysis code, LSENS. A major advantage of LSENS is the efficient procedures supporting systematic sensitivity analysis, which provides the basic methods for studying parameter sensitivities (i.e., changes in model behavior due to parameter variation). Sensitivity analysis establishes relationships between model predictions and problem parameters (i.e., initial concentrations, rate coefficients, etc). It helps determine the effects of uncertainties or changes in these input parameters on the predictions, which ultimately are compared with experimental observations in order to validate the model. Sensitivity analysis can identify parameters that must be determined accurately because of their large effect on the model predictions and parameters that need not be known with great precision because they have little or no effect on the solution. This capability may prove to be important in optimizing the design of experiments, thereby reducing the use of animals. This approach can be applied to study the metabolic effects of reduced oxygen delivery to cardiac muscle due to local myocardial ischemia and the effects of acute hypoxia on brain metabolism. Other important applications of sensitivity analysis include identification of quantitatively relevant pathways and biochemical species within an overall mechanism, when examining the effects of a genetic anomaly or pathological state on energetic system components and whole system behavior.

Radhakrishnan, Krishnan↗
NASA NTRS2000

Dietary fat intake, supplements, and weight loss

Although there remains controversy regarding the role of macronutrient balance in the etiology of obesity, the consumption of high-fat diets appears to be strongly implicated in its development. Evidence that fat oxidation does not adjust rapidly to acute increases in dietary fat, as well as a decreased capacity to oxidize fat in the postprandial state in the obese, suggest that diets high in fat may lead to the accumulation of fat stores. Novel data is also presented suggesting that in rodents, high-fat diets may lead to the development of leptin resistance in skeletal muscle and subsequent accumulations of muscle triacylglycerol. Nevertheless, several current fad diets recommend drastically reduced carbohydrate intake, with a concurrent increase in fat content. Such recommendations are based on the underlying assumption that by reducing circulating insulin levels, lipolysis and lipid oxidation will be enhanced and fat storage reduced. Numerous supplements are purported to increase fat oxidation (carnitine, conjugated linoleic acid), increase metabolic rate (ephedrine, pyruvate), or inhibit hepatic lipogenesis (hydroxycitrate). All of these compounds are currently marketed in supplemental form to increase weight loss, but few have actually been shown to be effective in scientific studies. To date, there is little or no evidence supporting that carnitine or hydroxycitrate supplementation are of any value for weight loss in humans. Supplements such as pyruvate have been shown to be effective at high dosages, but there is little mechanistic information to explain its purported effect or data to indicate its effectiveness at lower dosages. Conjugated linoleic acid has been shown to stimulate fat utilization and decrease body fat content in mice but has not been tested in humans. The effects of ephedrine, in conjunction with methylxanthines and aspirin, in humans appears unequivocal but includes various cardiovascular side effects. None of these compounds have been tested for their effectiveness or safety over prolonged periods of time.

NASA Discipline Musculoskeletal↗
NASA NTRS1993

Substrate oxidation capacity in rodent skeletal muscle: effects of exposure to zero gravity

A study was conducted, as part of the integrated National Aeronautics and Space Administration Space Life Sciences 1 mission flown in June of 1991, to ascertain the effects of 9 days of exposure to zero gravity on the capacity of rodent skeletal muscle fiber types to oxidize either [14C]pyruvate or [14C]palmitate under state 3 metabolic conditions, i.e., nonlimiting amounts of substrate and cofactors. In addition, activity levels of marker enzymes of the tricarboxylic acid cycle, malate shuttle, and beta-oxidation were measured. Results showed that significant differences in muscle weight occurred in both the predominantly slow vastus intermedius and predominantly fast vastus lateralis of flight vs. control groups (P < 0.05). Total protein content of the muscle samples was similar between groups. Both pyruvate oxidation capacity and the marker oxidative enzymes were not altered in the flight relative to control animals. However, the capacity to oxidize long-chain fatty acids was significantly reduced by 37% in both the high- and low-oxidative regions of the vastus muscle (P < 0.05). Although these findings of a selective reduction in fatty acid oxidation capacity in response to spaceflight are surprising, they are consistent with previous findings showing 1) an increased capacity to take up glucose and upregulate glucose transporter proteins and 2) a marked accumulation of triglycerides in the skeletal muscles of rats subjected to states of unloading. Thus, skeletal muscle of animals exposed to non-weight-bearing environments undergo subcellular transformations that may preferentially bias energy utilization to carbohydrates.

NASA Discipline Musculoskeletal↗
DOE OSTI2021

Microorganisms that co-consume glucose with non-glucose carbohydrates and methods of use

Microorganisms that co-consume glucose with non-glucose carbohydrates, such as xylose, and methods of using same. The microorganisms comprise modifications that reduce or ablate the activity of a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) protein or modifications that reduce or ablate the activity of a phosphoglucose isomerase and a GntR. The PTS protein may be selected from an enzyme I (EI), an HPr, an FPr, and an enzyme IIGlc (EIIGlc). Additional modifications include reduction or ablation of the activity of a pyruvate formate lyase, a lactate dehydrogenase, and a fumarate reductase and inclusion of recombinant pyruvate decarboxylase and alcohol dehydrogenase genes. The microorganisms are particularly suited to co-consuming glucose and xylose in media containing these substrates and producing ethanol therefrom.

Reed, Jennifer L.↗
DOE OSTI2024

Metabolic Engineering of Nonmodel Yeast Issatchenkia orientalis SD108 for 5–Aminolevulinic Acid Production

Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13 C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Here, our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Organic Acid Aerosol Measurements from the Mount Airy Site for CoURAGE

This study investigates the prevalence and distribution of organic acid aerosols in a rural environment using filter-based measurements collected in Mount Airy, Maryland, during the CoURAGE campaign from March 19th through June 12th 2025. PM2.5 filters quantify a range of organic acids commonly associated with secondary organic aerosol formation and atmospheric oxidation processes. Each filter was collected using a 15 LPM sampler and was extracted in ultrapure Millipore water (>18 MΩ), allowing water-soluble organic acids to be extracted into solution for analysis. The extracts were then examined using a Waters Acquity I-Class PLUS liquid chromatography system coupled to a Bruker Maxis-II ultra-high-resolution Q-TOF mass spectrometer with electrospray ionization, providing high-sensitivity detection and separation of target compounds. Concentrations of several key organic acids were quantified, including acetic, propionic, pyruvic, butyric, oxalic, isovaleric, valeric, malonic, maleic, succinic, glutaric, malic, adipic, and citric acids. These findings contribute to ongoing efforts to understand regional aerosol composition and their impacts on aerosol-cloud interactions.

Acetic acid↗
DOE OSTI2024

Toward Ultra‐High‐Quality‐Factor Wireless Masing Magnetic Resonance Sensing

It has recently been shown that a bolus of hyperpolarized nuclear spins can yield stimulated emission signals similar in nature to maser signals, potentially enabling new ways of sensing hyperpolarized contrast media, including most notably [1- 13 C]pyruvate that is under evaluation in over 50 clinical trials for metabolic imaging of cancer. The stimulated NMR signal emissions lasting for minutes do not require radio-frequency excitation, offering unprecedented advantages compared to conventional MR sensing. However, creating nuclear spin maser emission is challenging in practice due to stringent fundamental requirements, making practical in vivo applications hardly possible using conventional passive MR detectors. Here, in this study, we demonstrate the utility of a wireless NMR maser detector, the quality factor of which was enhanced 22-fold (to 1,670) via parametric pumping. This active-feedback technique breaks the intrinsic fundamental limit of NMR detector circuit quality factor. We show the use of parametric pumping to reduce the threshold requirement for inducing nuclear spin masing at 300 MHz resonance frequency in a preclinical MRI scanner. Indeed, stimulated emission from hyperpolarized protons was obtained under highly unfavorable conditions of low magnetic field homogeneity (T 2 * of 3 ms). Greater gains of the quality factor of the MR detector (up to 1 million) were also demonstrated.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

L–malic acid production from xylose by engineered Saccharomyces cerevisiae

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L –1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L –1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L –1 of malic acid was produced, with a productivity of 0.32 g L –1 h. Furthermore, these results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well!performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full alpha-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acety coA to alpha-santalene was compartmentalized into the mitochondria, leading to a 3.7 fold improvement in the production of alpha-santalene. Furthermore, the newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Thirdhand cigarette smoke leads to age-dependent and persistent alterations in the cecal microbiome of mice

The gut microbiome composition is influenced by many factors including environmental exposures. Here, we investigated the effect of thirdhand cigarette smoke (THS) and exposure age on gut microbiome diversity. C57BL/6 mice were exposed to THS at human exposure relevant levels for three weeks during three different life stages: postnatal (0–3 weeks of age), pubescent (4–7 weeks of age), and adult (9–12 weeks of age), respectively. Cecal microbiome profiles were assessed at 17 weeks of age by 16S rRNA gene sequencing. We found that age at THS exposure strongly influenced the cecal microbiome composition. Although postnatal THS exposure significantly influenced the microbial composition, pubescent and adulthood exposures only had minor effects. The microbiome of postnatally THS-exposed mice significantly increased several degradation pathways that regulate glycolysis and pyruvate decarboxylation, and significantly decreased coenzyme A biosynthesis and pyrimidine deoxyribonucleoside salvage. Our results indicate that mouse postnatal development is particularly susceptible to persistent THS exposure effects on the gut microbiome.

16S rRNA gene sequencing↗
DOE OSTI2021

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and applied it to screen 61eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functional competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Identification of a key water molecule involved in the macrophage migration inhibitory factor‐catalyzed tautomerization of para ‐hydroxyphenylpyruvate using neutron crystallography

Abstract Neutron crystallography was used to determine a 2.5‐Å resolution all‐atom structure of macrophage migration inhibitory factor (MIF) interacting with 3‐(4‐hydroxyphenyl)‐pyruvate (HPP). MIF is a pro‐inflammatory, pro‐tumorigenic protein that may be an attractive therapeutic target. MIF catalyzes the interconversion of the keto and enol forms of HPP by a tautomerase reaction. Although HPP is evidently not a physiological substrate of MIF, many compounds that inhibit this activity in enzymatic assays have been found also to inhibit physiological activities of MIF. Therefore, the MIF‐catalyzed HPP tautomerization reaction is used in initial screening of compounds in the search for inhibitors of MIF physiological activity. The neutron diffraction‐derived crystal structure reveals the position of a water molecule involved in the tautomerization reaction, and also confirms the charged state of lysine‐32 in the active site. The structure confirms the previously proposed catalytic mechanism of MIF, with the N‐terminal Pro‐1 abstracting a proton to generate an HPP enolate intermediate which is subsequently protonated. The structure reported herein reveals that this proton is supplied by a neighboring water molecule. Along with the neutron structure, a room‐temperature synchrotron x‐ray crystal structure reveals a covalent adduct between HPP and MIF. While this adduct is a result of radiation‐induced chemistry, its formation confirms the catalytic role of the active site residue because a covalent complex could only form if the reactive carbon of the substrate is correctly positioned by the enzyme.

Schröder, Gabriela C. [Department of Molecular and↗