Browsing by Subject "Catalysis"

Now showing 1 - 9 of 9
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    A Molecular Mechanism for Autoinhibition of Myosin Light Chain Kinases
    (Elsevier, 1993) Gallagher, Patricia J.; Herring, B. Paul; Trafny, Andrzej; Sowadski, Janusz; Stull, James T.; Anatomy, Cell Biology and Physiology, School of Medicine
    It is postulated that basic residues within the inhibitory region of myosin light chain kinase (MLCK) bind acidic residues within the catalytic core to maintain the kinase in an inactive form. In this study, we identified residues within the catalytic cores of the skeletal and smooth muscle MLCKs that may bind basic residues in inhibitory region. Acidic residues within the catalytic core of the rabbit skeletal and smooth muscle MLCKs were mutated and the kinetic properties of the mutant kinases determined. Mutation of 6 and 8 acidic residues in the skeletal and smooth muscle MLCKs, respectively, result in mutant MLCKs with decreases in KCaM (the concentration of calmodulin required for half-maximal activation of myosin light chain kinase) value ranging from 2- to 100-fold. Two inhibitory domain binding residues identified in each kinase also bind a basic residue in light chain substrate. The remaining mutants all have wild-type Km values for light chain. The predicted inhibitory domain binding residues are distributed in a linear fashion across the surface of the lower lobe of the proposed molecular model of the smooth muscle MLCK catalytic core. As 6 of the inhibitory domain binding residues in the smooth muscle MLCK are conserved in other Ca2+/calmodulin-dependent protein kinases, the structural basis for autoinhibition and activation may be similar.
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    A Radical Transfer Pathway in Spore Photoproduct Lyase
    (ACS, 2013) Yang, Linlin; Nelson, Renae S.; Benjdia, Alhosna; Lin, Gengjie; Telser, Joshua; Stoll, Stefan; Schlichting, Ilme; Li, Lei; Chemistry and Chemical Biology, School of Science
    Spore photoproduct lyase (SPL) repairs a covalent UV-induced thymine dimer, spore photoproduct (SP), in germinating endospores and is responsible for the strong UV resistance of endospores. SPL is a radical S-adenosyl-l-methionine (SAM) enzyme, which uses a [4Fe-4S](+) cluster to reduce SAM, generating a catalytic 5'-deoxyadenosyl radical (5'-dA(•)). This in turn abstracts a H atom from SP, generating an SP radical that undergoes β scission to form a repaired 5'-thymine and a 3'-thymine allylic radical. Recent biochemical and structural data suggest that a conserved cysteine donates a H atom to the thymine radical, resulting in a putative thiyl radical. Here we present structural and biochemical data that suggest that two conserved tyrosines are also critical in enzyme catalysis. One [Y99(Bs) in Bacillus subtilis SPL] is downstream of the cysteine, suggesting that SPL uses a novel hydrogen atom transfer (HAT) pathway with a pair of cysteine and tyrosine residues to regenerate SAM. The other tyrosine [Y97(Bs)] has a structural role to facilitate SAM binding; it may also contribute to the SAM regeneration process by interacting with the putative (•)Y99(Bs) and/or 5'-dA(•) intermediates to lower the energy barrier for the second H abstraction step. Our results indicate that SPL is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating HAT chain.
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    Investigation of the potential bacterial proteasome homologue Anbu
    (2014-09-08) Suknaic, Stephen R.; Kusmierczyk, Andrew; Randall, Stephen Karl, 1953-; Anderson, Gregory G.
    Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.
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    Kinetic Analysis of Primate and Ancestral Alcohol Dehydrogenases
    (2012-11-29) Myers, Candace R.; Hurley, Thomas D., 1961-; Goebl, Mark G.; Mosley, Amber L.
    Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.
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    Oxidation of Hydrogen Sulfide to Polysulfide and Thiosulfate by a Carbon Nanozyme: Therapeutic Implications with an Emphasis on Down Syndrome
    (Wiley, 2024) Derry, Paul J.; Liopo, Anton V.; Mouli, Karthik; McHugh, Emily A.; Vo, Anh T. T.; McKelvey, Ann; Suv, Larry J.; Wu, Gang; Gao, Yan; Olson, Kenneth R.; Tour, James M.; Kent, Thomas A.; Cellular and Integrative Physiology, School of Medicine
    Hydrogen sulfide (H2 S) is a noxious, potentially poisonous, but necessary gas produced from sulfur metabolism in humans. In Down Syndrome (DS), the production of H2 S is elevated and associated with degraded mitochondrial function. Therefore, removing H2 S from the body as a stable oxide could be an approach to reducing the deleterious effects of H2 S in DS. In this report we describe the catalytic oxidation of hydrogen sulfide (H2 S) to polysulfides (HS2+n - ) and thiosulfate (S2 O3 2- ) by poly(ethylene glycol) hydrophilic carbon clusters (PEG-HCCs) and poly(ethylene glycol) oxidized activated charcoal (PEG-OACs), examples of oxidized carbon nanozymes (OCNs). We show that OCNs oxidize H2 S to polysulfides and S2 O3 2- in a dose-dependent manner. The reaction is dependent on O2 and the presence of quinone groups on the OCNs. In DS donor lymphocytes we found that OCNs increased polysulfide production, proliferation, and afforded protection against additional toxic levels of H2 S compared to untreated DS lymphocytes. Finally, in Dp16 and Ts65DN murine models of DS, we found that OCNs restored osteoclast differentiation. This new action suggests potential facile translation into the clinic for conditions involving excess H2 S exemplified by DS.
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    Probing the mechanism of Bacillus subtilis oxalate decarboxylase
    (2015-12-01) Zhu, Wen; Richards, Nigel G.; Li, Lei; Shah, Kavita; Mesecar, Andrew; Long, Eric C.
    Oxalate decarboxylase (EC 4. 1. 1. 2 OxDC) from Bacillus subtilis is a manganese-dependent enzyme that catalyzes the cleavage of the chemically inactive C-C bond in oxalate to yield formate and carbon dioxide. A mechanism involving Mn(III) has been proposed for OxDC, however no clear spectroscopic evidence to support this mechanism has yet been obtained. In addition, a recent study has shown that N-terminal metal binding site loop variants of OxDC were able to catalyze the oxidation of oxalate to yield hydrogen peroxide and carbon dioxide, which makes OxDc function as another oxalate degradation protein in the cupin superfamily, oxalate oxidase (EC 1.2.3.4 OxOx). In this work, wild-type (WT) Bacillus subtilis OxDC and a series of variants with mutations on conserved residues were characterized to investigate the catalytic mechanism of OxDC. The application of membrane inlet mass spectrometry (MIMS), electronic paramagnetic resonance (EPR) spectroscopy and kinetic isotope effects (KIEs) provided information about the mechanism. The Mn(III) was identified and characterized under acidic conditions in the presence of dioxygen and oxalate. Mutations on the second shell residues in the N-terminal metal binding site affected the enzyme activity properties of the metal. In the N-terminal domain, the functional importance of the residues in the active site loop region, especially Glu162, was confirmed, and evidence for the previously proposed mechanism in which OxDC and the OxDC/OxOx chimeric variant share the initial steps has been found. In addition, the mono-dentate coordination of oxalate in the N-terminal metal binding site was confirmed by X-ray crystallography. A proteinase cleavable OxDC was constructed and characterized, revealing the interaction between the N-terminal and C-terminal domains.
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    Topical ferumoxytol nanoparticles disrupt biofilms and prevent tooth decay in vivo via intrinsic catalytic activity
    (Springer Nature, 2018-07-31) Liu, Yuan; Naha, Pratap C.; Hwang, Geelsu; Kim, Dongyeop; Huang, Yue; Simon-Soro, Aurea; Jung, Hoi-In; Ren, Zhi; Li, Yong; Gubara, Sarah; Alawi, Faizan; Zero, Domenick; Hara, Anderson T.; Cormode, David P.; Cariology, Operative Dentistry and Dental Public Health, School of Dentistry
    Ferumoxytol is a nanoparticle formulation approved by the U.S. Food and Drug Administration for systemic use to treat iron deficiency. Here, we show that, in addition, ferumoxytol disrupts intractable oral biofilms and prevents tooth decay (dental caries) via intrinsic peroxidase-like activity. Ferumoxytol binds within the biofilm ultrastructure and generates free radicals from hydrogen peroxide (H2O2), causing in situ bacterial death via cell membrane disruption and extracellular polymeric substances matrix degradation. In combination with low concentrations of H2O2, ferumoxytol inhibits biofilm accumulation on natural teeth in a human-derived ex vivo biofilm model, and prevents acid damage of the mineralized tissue. Topical oral treatment with ferumoxytol and H2O2 suppresses the development of dental caries in vivo, preventing the onset of severe tooth decay (cavities) in a rodent model of the disease. Microbiome and histological analyses show no adverse effects on oral microbiota diversity, and gingival and mucosal tissues. Our results reveal a new biomedical application for ferumoxytol as topical treatment of a prevalent and costly biofilm-induced oral disease.
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    Two-Dimensional Titanium and Molybdenum Carbide MXenes as Electrocatalysts for CO2 Reduction
    (Elsevier, 2020-06-26) Handoko, Albertus D.; Chen, Hetian; Lum, Yanwei; Zhang, Qianfan; Anasori, Babak; Seh, Zhi Wei; Mechanical Engineering and Energy, School of Engineering and Technology
    Electrocatalytic CO2 reduction reaction (CO2RR) is an attractive way to produce renewable fuel and chemical feedstock, especially when coupled with efficient CO2 capture and clean energy sources. On the fundamental side, research on improving CO2RR activity still revolves around late transition metal-based catalysts, which are limited by unfavorable scaling relations despite intense investigation. Here, we report a combined experimental and theoretical investigation into electrocatalytic CO2RR on Ti- and Mo-based MXene catalysts. Formic acid is found as the main product on Ti2CTx and Mo2CTx MXenes, with peak Faradaic efficiency of over 56% on Ti2CTx and partial current density of up to −2.5 mA cm−2 on Mo2CTx. Furthermore, simulations reveal the critical role of the Tx group: a smaller overpotential is found to occur at lower amounts of –F termination. This work represents an important step toward experimental demonstration of MXenes for more complex electrocatalytic reactions in the future.