Hyper-reactive Cysteines Research Articles

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

HyperCys: A Structure- and Sequence-Based Predictor of Hyper-Reactive Druggable Cysteines.

The cysteine side chain has a free thiol group, making it the amino acid residue most often covalently modified by small molecules possessing weakly electrophilic warheads, thereby prolonging on-target residence time and reducing the risk of idiosyncratic drug toxicity. However, not all cysteines are equally reactive or accessible. Hence, to identify targetable cysteines, we propose a novel ensemble stacked machine learning (ML) model to predict hyper-reactive druggable cysteines, named HyperCys. First, the pocket, conservation, structural and energy profiles, and physicochemical properties of (non)covalently bound cysteines were collected from both protein sequences and 3D structures of protein-ligand complexes. Then, we established the HyperCys ensemble stacked model by integrating six different ML models, including K-nearest neighbors, support vector machine, light gradient boost machine, multi-layer perceptron classifier, random forest, and the meta-classifier model logistic regression. Finally, based on the hyper-reactive cysteines' classification accuracy and other metrics, the results for different feature group combinations were compared. The results show that the accuracy, F1 score, recall score, and ROC AUC values of HyperCys are 0.784, 0.754, 0.742, and 0.824, respectively, after performing 10-fold CV with the best window size. Compared to traditional ML models with only sequenced-based features or only 3D structural features, HyperCys is more accurate at predicting hyper-reactive druggable cysteines. It is anticipated that HyperCys will be an effective tool for discovering new potential reactive cysteines in a wide range of nucleophilic proteins and will provide an important contribution to the design of targeted covalent inhibitors with high potency and selectivity.

Proteomics characterization of mitochondrial-derived vesicles under oxidative stress.

Mitochondria share attributes of vesicular transport with their bacterial ancestors given their ability to form mitochondrial‐derived vesicles (MDVs). MDVs are involved in mitochondrial quality control and their formation is enhanced with stress and may, therefore, play a potential role in mitochondrial‐cellular communication. However, MDV proteomic cargo has remained mostly undefined. In this study, we strategically used an in vitro MDV budding/reconstitution assay on cardiac mitochondria, followed by graded oxidative stress, to identify and characterize the MDV proteome. Our results confirmed previously identified cardiac MDV markers, while also revealing a complete map of the MDV proteome, paving the way to a better understanding of the role of MDVs. The oxidative stress vulnerability of proteins directed the cargo loading of MDVs, which was enhanced by antimycin A (Ant‐A). Among OXPHOS complexes, complexes III and V were found to be Ant‐A‐sensitive. Proteins from metabolic pathways such as the TCA cycle and fatty acid metabolism, along with Fe‐S cluster, antioxidant response proteins, and autophagy were also found to be Ant‐A sensitive. Intriguingly, proteins containing hyper‐reactive cysteine residues, metabolic redox switches, including professional redox enzymes and those that mediate iron metabolism, were found to be components of MDV cargo with Ant‐A sensitivity. Last, we revealed a possible contribution of MDVs to the formation of extracellular vesicles, which may indicate mitochondrial stress. In conclusion, our study provides an MDV proteomics signature that delineates MDV cargo selectivity and hints at the potential for MDVs and their novel protein cargo to serve as vital biomarkers during mitochondrial stress and related pathologies.

Sequence-Based Prediction of Cysteine Reactivity Using Machine Learning

As one of the most intrinsically reactive amino acids, cysteine carries a variety of important biochemical functions, including catalysis and redox regulation. Discovery and characterization of cysteines with heightened reactivity will help annotate protein functions. Chemical proteomic methods have been used to quantitatively profile cysteine reactivity in native proteomes, showing a strong correlation between the chemical reactivity of a cysteine and its functionality; however, the relationship between the cysteine reactivity and its local sequence has not yet been systematically explored. Herein, we report a machine learning method, sbPCR (sequence-based prediction of cysteine reactivity), which combines the basic local alignment search tool, truncated composition of k-spaced amino acid pair analysis, and support vector machine to predict cysteines with hyper-reactivity based on only local sequence features. Using a benchmark set compiled from hyper-reactive cysteines in human proteomes, our method can achieve a prediction accuracy of 98%, a precision of 95%, and a recall ratio of 89%. We utilized these governing features of local sequence motifs to expand the prediction to potential hyper-reactive cysteines in other proteomes deposited in the UniProt database. We validated our predictions in Escherichia coli by activity-based protein profiling and discovered a hyper-reactive cysteine from a functionally uncharacterized protein, YecH. Biochemical analysis suggests that the hyper-reactive cysteine might be involved in metal binding. Our computational method provides a large inventory of potential hyper-reactive cysteines in proteomes and is highly complementary to other experimental approaches to guidesystematic annotation of protein functions in the postgenome era.

Identifying Functional Cysteine Residues in the Mitochondria.

The mitochondria are dynamic organelles that regulate oxidative metabolism and mediate cellular redox homeostasis. Proteins within the mitochondria are exposed to large fluxes in the surrounding redox environment. In particular, cysteine residues within mitochondrial proteins sense and respond to these redox changes through oxidative modifications of the cysteine thiol group. These oxidative modifications result in a loss in cysteine reactivity, which can be monitored using cysteine-reactive chemical probes and quantitative mass spectrometry (MS). Analysis of cell lysates treated with cysteine-reactive probes enable the identification of hundreds of cysteine residues, however, the mitochondrial proteome is poorly represented (<10% of identified peptides), due to the low abundance of mitochondrial proteins and suppression of mitochondrial peptide MS signals by highly abundant cytosolic peptides. Here, we apply a mitochondrial isolation and purification protocol to substantially increase coverage of the mitochondrial cysteine proteome. Over 1500 cysteine residues from ∼450 mitochondrial proteins were identified, thereby enabling interrogation of an unprecedented number of mitochondrial cysteines. Specifically, these mitochondrial cysteines were ranked by reactivity to identify hyper-reactive cysteines with potential catalytic and regulatory functional roles. Furthermore, analyses of mitochondria exposed to nitrosative stress revealed previously uncharacterized sites of protein S-nitrosation on mitochondrial proteins. Together, the mitochondrial cysteine enrichment strategy presented herein enables detailed characterization of protein modifications that occur within the mitochondria during (patho)physiological fluxes in the redox environment.

The extreme hyper-reactivity of selected cysteines drives hierarchical disulfide bond formation in serum albumin.

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.

Novel Inhibitors for PRMT1 Discovered by High-Throughput Screening Using Activity-Based Fluorescence Polarization

Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high-throughput screening assay for PRMT1 that utilizes a hyper-reactive cysteine within the active site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine-containing cysteine-reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM-dependent methyltransferases.

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Quantitative reactivity profiling predicts functional cysteines in proteomes

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here, we describe a proteomics method to quantitatively profile the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyperreactivity was a rare feature among cysteines and found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyperreactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and involved in iron-sulfur protein biogenesis. Finally, we demonstrate that quantitative reactivity profiling can also form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

Reactive Cysteines of Ryanodine Receptor Type 1 Influence Function and Response to Oxidative Stress

Redox modulation of the skeletal muscle ryanodine receptor1 (RyR1) plays a key role in determining the responsiveness of the Ca2+ release channel to physiological modulation. The sensitivity of RyR1 to redox stress may be conferred by seven previously identified hyper-reactive cysteines (1040, 1303, 2426, 2606, 2611, 2625 and 3635). Wild type RyR1 (wtRyR1), and seven hyper-reactive cysteine mutations of RyR1 were stably expressed in HEK-293 cells and their contribution to RyR1 function evaluated. Addition of RyR1 activator 4-chloro-m-cresol (4-CMC) elicited an increase in [Ca2+]i in the wtRyR1cells but failed to produce a Ca2+ response in the C1303S, C2606S, C2436S and C7S (expressing all seven cysteine mutations) expressing cells, while the C1040S and C2611S mutations significantly attenuated 4-CMC mediated Ca2+ response. Microsomal fractions isolated from C1040S, C2611S, C2436S and C3635S bound to 3[H]Ry while C7S and C1303S showed significantly lower, levels of RyR-binding, although significantly above preparations from RyR-null HEK 293. The sensitivity of RyR1 to 1, 4-napthoquinone (NQ) appears to depend on the expression of RyR1 and the presence of reactive cysteines. Sensitivity to NQ-induced cytotoxicity was determined by the multi-Tox fluorescence assay. NQ decreased cell viability in a dose-dependent manner, but the wtRyR1 cells were less sensitive than C2606, C1040S, C2611S and the C7S mutants. These data indicate the role of hyper-reactive cysteines in regulating RyR1 function and its response to oxidative stress. Supported by NIH AR43140.

Ryanodine Receptor Structure: Progress and Challenges

Ryanodine Receptor Structure: Progress and Challenges

Conformation-dependent Stability of Junctophilin 1 (JP1) and Ryanodine Receptor Type 1 (RyR1) Channel Complex Is Mediated by Their Hyper-reactive Thiols

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.

Structural Insights into the Mechanism of PEPCK Catalysis,

Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Identification of Hyperreactive Cysteines within Ryanodine Receptor Type 1 by Mass Spectrometry

The skeletal-type ryanodine receptor (RyR1) undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and non-covalent interactions driven by the transmembrane redox potential that enable redox sensing. Tight redox regulation of RyR1 is thought to be primarily mediated through highly reactive (hyperreactive) cysteines. Of the 100 cysteines per subunit of RyR1, approximately 25-50 are reduced, with 6-8 considered hyperreactive. Thus far, only Cys-3635, which undergoes selective adduction by NO, has been identified. In this report, RyR1-enriched junctional sarcoplasmic reticulum is labeled with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, 1 pmol/microg of protein) in the presence of 10 mm Mg(2+), conditions previously shown to selectively label hyperreactive sulfhydryls and eliminate redox sensing. The CPM-adducted RyR1 is separated by gel electrophoresis and subjected to in-gel tryptic digestion. Isolation of CPM-adducted peptides is achieved by analytical and microbore high-performance liquid chromatography utilizing fluorescence and UV detection. Subsequent analysis using two direct and one tandem mass spectrometry methods results in peptide masses and sequence data that, compared with the known primary sequence of RyR1, enable unequivocal identification of CPM-adducted cysteines. This work is the first to directly identify seven hyperreactive cysteines: 1040, 1303, 2436, 2565, 2606, 2611, and 3635 of RyR1. In addition to Cys-3635, the nitrosylation site, six additional cysteines may contribute toward redox regulation of the RyR1 complex.

Regulation of aldose reductase and the polyol pathway activity by nitric oxide

Increased flux of glucose through the polyol pathway has been implicated in the pathophysiology of secondary diabetic complications. The first step of this pathway, which generates sorbitol from glucose, is catalyzed by aldose reductase (AR) (AKR1B). In vitro, the binding of substrates and inhibitors to AR is highly sensitive to the oxidation state of the enzyme due to the presence of a hyper-reactive cysteine residue at the active site of the enzyme. This residue (Cys-298) can be readily modified in air or, by thiol-modifying reagents, nitric oxide (NO) donors and nitrosothiols. We show that exposure of rat erythrocytes to NO donors inhibits AR activity and AR mediated accumulation of sorbitol, possibly by S-glutathiolation of Cys-298. Both glutathiolation and inhibition of AR are reversible upon subsequent incubation of the cells with fresh media without NO donors. These observations suggest that NO regulates the cellular activity of AR and in turn the flux of glucose via the polyol pathway. The inhibition of AR by exogenous or endogenous NO appears to be related to reversible S-glutathiolation of the AR protein. Because hyperglycemic states are associated with a decrease in NO generation, the loss of NO-mediated repression of AR may be a significant factor in the activation of the polyol pathway and the development of secondary diabetic complications.

Functional role of hyperreactive sulfhydryl moieties within the ryanodine receptor complex.

Several laboratories using chemically heterogeneous sulfhydryl modifying agents have shown that sarcoplasmic reticulum (SR) Ca2+ channels known as ryanodine receptors (RyRs) are especially sensitive to modification of functionally important cysteine residues. The functional consequence of sulfhydryl modification of RyRs can include phases of activation and inhibition that are very much dependent on the concentration of the reagent used, the length of exposure, and the nature of the chemical reaction the reagent undertakes with sulfhydryl groups. Most challenging is understanding the relationship for how specific sulfhydryl moieties ascribe specific aspects of RyR function. Considering the structural complexity of the RyR complex with its associated proteins, this task is likely to be a formidable one. A small number of hyperreactive thiols have been shown to exist within the RyR complex. Their functional role does not appear to impact directly on channel gating. Rather hyperreactive cysteine (Cys) moieties may represent biochemical components of a redox sensor that conveys information about localized changes in redox potential produced by physiologic (e.g., glutathione, nitric oxide) and pathophysiologic (quinones, reactive oxygen species) channel modulators to the Ca2+ release process. The molecular and functional details of such a redox sensor remains to be elucidated.

Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phospho enolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg 2+ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co 2+ > Mn 2+ > Cd u2+ > Ni 2+ ⪢ Fe 2+ > VO 2+, while Zn 2+ and Ca 2+ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO 2 substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn 2+ activated enzyme the inhibition was purely non-competitive ( K ii = K is) towards all substrates, while for the Co 2+-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn 2+-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO 3 − had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co 2+-activated enzyme than for its Mn 2+-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn 2+ and ADP afforded almost complete protection from inactivation; in the presence of Co 2+ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.

Exploring the catalytic mechanism of skeletal muscle udp-glucose pyrophosphorylase: Identification of a hyperreactive cysteine at the enzyme active site

1. 1. The involvement of cysteine residues in the catalytic mechanism of UDP-glucose pyrophosphorylase was suggested by the rapid inactivation of the enzyme by N-ethylmaleimide, even at 1:1 reagent/enzyme stoichiometric ratios. 2. 2. The inactivation is largely prevented by uridine substrates (UDP-glucose and UTP) in agreement with the assumption that the reactive cysteine is located at the active site.