Real‐time measurements of dark substrate catalysis

Xie, Donghua; Suvorov, Leonid I.; Erickson, John W.; Gulnik, Sergei V.

doi:10.1110/ps.8.11.2460

Cited by 12 publications

(5 citation statements)

References 18 publications

(14 reference statements)

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“…Competition assays with peptides were performed at 200 μM 4MU-GlcNAc. K m determination for GlcNAc-modified peptides was performed following an approach using established equations for multisubstrate enzyme kinetics to derive kinetic constants of a substrate whose turnover cannot be detected directly [ 24 ]. The fluorigenic reporter substrate and the GlcNAcylated peptide are considered as competitive inhibitors of each other.…”

Section: Methodsmentioning

confidence: 99%

Human OGA binds substrates in a conserved peptide recognition groove

Schimpl

Schüttelkopf

Borodkin

et al. 2010

Biochemical Journal

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Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.

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Section: Methodsmentioning

confidence: 99%

Human OGA binds substrates in a conserved peptide recognition groove

Schimpl

Schüttelkopf

Borodkin

et al. 2010

Biochemical Journal

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“…The initial rate of interaction of Red-Gal substrate with β -galactosidase enzyme can be described by the Michaelis-Menten equation [ 26 ], where, k cat is turnover number and E o is concentration of β -galactosidase enzyme (released from E . coli bacteria), K m is Michaelis constant and S is the concentration of Red-Gal substrate.…”

Section: Resultsmentioning

confidence: 99%

DipTest: A litmus test for E. coli detection in water

2017

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We have developed a new litmus paper test (DipTest) for detecting Escherichia coli (E. coli) in water samples by performing enzymatic reactions directly on the porous paper substrate. The paper strip consists of a long narrow piece of cellulose blotting paper coated with chemoattractant (at bottom edge), wax hydrophobic barrier (at the top edge), and custom formulated chemical reagents (at reaction zone immediately below the wax hydrophobic barrier). When the paper strip is dipped in water, E. coli in the water sample is attracted toward the paper strip due to a chemotaxic mechanism followed by the ascent along the paper strip toward the reaction zone due to a capillary wicking mechanism, and finally the capillary motion is arrested at the top edge of the paper strip by the hydrophobic barrier. The E. coli concentrated at the reaction zone of the paper strip will react with custom formulated chemical reagents to produce a pinkish-red color. Such a color change on the paper strip when dipped into water samples indicates the presence of E. coli contamination in potable water. The performance of the DipTest device has been checked with different known concentrations of E. coli contaminated water samples using different dip and wait times. The DipTest device has also been tested with different interfering bacteria and chemical contaminants. It has been observed that the different interfering contaminants do not have any impact on the DipTest, and it can become a potential solution for screening water samples for E. coli contamination at the point of source.

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“…Monkey kidney COS-1 cells were selected for these experiments as they provide a favorable metabolic background. COS-1 cells were transfected with the AKR1C2 plasmid, and the required kinetic parameters were obtained by measuring the effect of each steroid on the Coumberone metabolism rate in intact cells using the competition assay between the reporter substrate and the dark substrate (Figure ) − . Importantly, the apparent K i value of testosterone was 8-fold higher than the value of the apparent K m of DHT ( K i(T) = 8.7 ± 1.8 μM; K m(DHT) = 1.1 ± 0.1 μM).…”

Section: Resultsmentioning

confidence: 99%

“…To compare the effects of the competitive “dark” substrate (DHT), its precursor (testosterone), and its metabolite (3α-diol), on the metabolism of Coumberone, the probe metabolism rate was related to the metabolic rate in the presence of the different steroids . These relative rates were then fit to the equation ν i /ν 0 = [ (1+ K M / S ) ]/[1 + K M / S (1 + S ′/ K ′ M ) ] by using KaleidaGraph to yield K ′ M of DHT (the Michaelis constant for the dark substrate) as well as the K ′ i of the ligands (testosterone and 3α-diol) as described .…”

Section: Methodsmentioning

confidence: 99%

Expanding the Use of Fluorogenic Enzyme Reporter Substrates to Imaging Metabolic Flux Changes: the Activity Measurement of 5α-Steroid Reductase in Intact Mammalian Cells

2010

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The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5α-reductase to DHT (5α-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17β-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5α-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5α-R1 (5α-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5α-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.

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Real‐time measurements of dark substrate catalysis

Cited by 12 publications

References 18 publications

Human OGA binds substrates in a conserved peptide recognition groove

Human OGA binds substrates in a conserved peptide recognition groove

DipTest: A litmus test for E. coli detection in water

Expanding the Use of Fluorogenic Enzyme Reporter Substrates to Imaging Metabolic Flux Changes: the Activity Measurement of 5α-Steroid Reductase in Intact Mammalian Cells

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