Microsoft Word Eu(III) Enzyme Assay ESI Submitted.doc

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1 for (ESI) Material Supplementary Electronic Chemical Science. 2019 This journal is © The Royal Society of Chemistry Supporting Information A simple, robust, universal assay for real - time enzyme monitoring by signaling changes in nucleoside phosphate anion concentration based anion receptor - using a europium(III) † † † † , Rozee Ali , Charlotte A. Antonen , Chloe Sarah H. Hewitt , Romain Mailhot † ‡ Dodson and Stephen J. Butler * † Department of Chemistry, Loughborough University, Epinal Way, Loughborough, LE11 3TU, UK. ‡ Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK. 1

2 General Cons iderations All chemicals used were purchased from standard chemical suppliers and used without further 1 + [ ] Eu.1 purification. was synthesised using a synthetic protocol described previously. Measurements of pH were carried out using a Jenway 3510 pH/mV me ter with a Jenway combination electrode or a Jenway 3020 pH meter with an Aldrich glass combination pH electrode, both calibrated using buffer solutions of pH 4.00 ± 0.01, 7.00 ± 0.01 and 10.00 ± 0.01. 1.1 Optical Spectroscopy e measured using a UV -­‐ 1800 UV -­‐ spectrophotometer. Shimadzu UV/Vis absorbance spectra wer F luoromax luminescence spectrometer using dM300 Emission spectra were recorded on a SPEX version 3.12 software. Emission spectra were obtained using a 40 μL Hellma Analytics quartz K 111 -­‐ 10 -­‐ -­‐ cuvette (Art no. 40). E xcitation light was set a t 330 nm, and emission read in the range 550 – 720 nm (for titrations) or 570 – 635 nm (for enzyme assays), using an integration and emission slits of 0.5 nm. time of 0.5 seconds, increment of 0.5 nm and excitation Plate reader data was obtained on a BMG La btech CLARIOstar microplate reader in black Fisherbrand 384 -­‐ well plates , using a total volume of 40 μL per well. ™ 1.2 Biophysical measurement procedures All biophysical analyses were carried out in degassed 10 mM HEPES, pH 7.0 buffer , unless 1 + -­‐ otherwise stated [ Eu.1 ] . Concentrated stocks were made at ~2.5 mg mL prior to in methanol diluting to 80 μM in 10 mM HEPES, pH 7.0 and the accurate concentration determined using the -­‐ 1 -­‐ 1 1 0 M Unless otherwise ) UV/Vis absorbance at 332 nm (ε = 12 50 in 10 mM HEPES, pH 7.0. cm + [ Eu.1 ] stated, concentration was kept at 8 μM concentration. Stocks of phosphoanions were the made up at 5 or 2 5 mM and adjusted to pH 7.0 by addition of minimal volumes of 1 M NaOH or 1 M HCl . Stocks of MgCl Stocks and DTT were made up at 50 or 250 mM in 10 mM HEPEs, pH 7.0. 2 of sugars (glucose, maltose, mannose, fructose and lactose) were made up at 100 mM. Anion screen + of 50 μL of [ Eu.1 were Emission spectra (8 μM) with the phosphoanions (1 mM) , or b uffer ] taken . 1 μL of 250 mM MgCl was added, and the emission spectrum taken again. 2 2

3 Fluorometer based enzyme simulation -­‐ solution of Variable amounts of a product, containing the phosphoanion n appropriate enzyme + ( generally 5 mM) (generally 1 mM), MgCl 8 μM [ Eu.1 ] in 10 , and any other stated components , 2 mM HEPES (pH 7.0) was added to 50 μL of enzymatic substrate , containing the a solution of + (generally 5 mM), MgCl Eu.1 ] phosphoanion (generally 1 mM), (8 μM) and any other stated [ 2 in 10 mM HEPES (pH 7.0) . E mission spectra (λ = 330 nm, λ – = 550 components 720 nm) em exc were recorded after each addition. The ratio of the intensity at 616.5 nm to 599.5 nm (or s and plotted against the percentage of enzyme product in the cuvette. ) w ere stated taken Plate -­‐ based enzyme simulation D iffering ratios of a solution of enzyme product or substrate, containing the phosphoanion + generally (generally 5 mM) , [ Eu.1 ] (generally 1 mM), MgCl ( and other state components 8 μM) 2 in 10 mM HEPES (pH 7.0) to a total volume of 40 μL were added to a 384 -­‐ w ell plate , in triplicate, taken and the plate incubated for 30 minutes prior to reading. Luminescence intensities were an d with excitation at 292 – 366 nm 625 emission using time -­‐ resolved measurements at 615 – nm using an integration time of 60 – 400 μ s or intensity at 601 -­‐ 6 33 nm. The mean of the triplicate intensity values were taken and plotted against the percentage enzyme product in the well. Error bars indicate the standard error in mean . General h exokinase enzyme assay To 20 μL of a solution containi ng ATP (2 mM or 2 ⨯ final concentration ) , MgCl (10 mM or 2 ⨯ 2 + ) , [Eu.1] (16 μM final concentration or 2 ⨯ final concentration ) and stated other components (at final concentration) 2 ⨯ in 10 mM HEPES, pH 7.0 in a 384 -­‐ well plate was added 10 μ L of a solution o f hexokinase (at 4 ⨯ final concentration) or buffer , and the plate incubated for 15 f glucose (or appropriate other , at 4 ⨯ minutes. 10 μL of a inal concentration ) was added sugar and the plate read immediately. Luminescence intensities were taken with excit ation at 292 – 366 nm and emission using time -­‐ resolved measurements at 615 – 625 nm using an integration time of 60 – 400 μs or intensity at 601 -­‐ 633 nm , at appropriate time intervals (often 30 s) . Assays were run in triplicate and t he mean of the triplicat e intensity values were taken and plotted against time . Where appropriate bleaching (no enzyme) experiments were taken away hexokinase. from experiments containing 3

4 Hexokinase different concentrations of ATP MgCl , or fructose 2 A titration using a tion regime was used to vary the concentrations, the protocol is 2/3 dilu detailed below. to 16 wells in one row of a 384 -­‐ well plate. 4 0 μL of an appropriate 20 μL of buffer was added or fructose solution was added to the first well and mixed. 40 μL was transferred ATP, MgCl 2 th peated to the 15 well , from the first well to the second well and mixed. This process was re th the 16 10 μL of a solution where the 40 μL was discarded, and well left with just buffer. + if appropriate ⨯ final concentration) , 32 μM [Eu .1] containing , MgCl (20 mM hexokinase (at 4 ) 2 was added to e ATP ( 4 mM if appropriate) and ach well and the plate incubated for 15 minutes. 10 μL of a solution of ATP (4 mM) or glucose ( 40 mM) as appropriate was added and the plate and read immediately . Luminescence intensities were taken with excitation at 292 – 366 nm reso lved measurements at 615 emission using time -­‐ – – 625 nm using an integration time of 60 400 μs or at appropriate time intervals (often 30 s). Assays were run in triplicate and the mean of the triplicate intensity values were taken and plotted against time. fructose M ichaelis Menten calculations the initial rate was calculated as the gradient of For the -­‐ 5 for each measurement . These initial rates the intensity against time over the first minutes a Michaelis -­‐ Menten equation were plotted against fructose concentration and fit to using Origin 2015. Aurora A enzyme assay 50 mM NaCl, 5 mM MgCl Aurora A enzyme assays were performed in a buffer containing , 0.25 2 mM DTT, 2.5 % glycerol in 10 mM HEPES, pH 7.0. To a 20 μL solution containing kemptide (2 + and [Eu.1] mM) (16 μM) was added 10 μL of an appropriate Aurora A solution in a 384 -­‐ well plate. The wells were mixed a the plate incubated for 15 minutes. 10 μL of ATP (4 mM) was nd added and the plate read immed iately. Luminescence intensities were taken with excitation at 292 – 366 nm and emission using time -­‐ resolved measurements at 615 – 625 nm using an integration time of 60 400 μs, at appropriate time intervals . Assays were run in triplicate and – the mean of the triplicate intensity values were taken and plotted against time. Where appropriate bleaching (no enzyme) experiments were taken away from experiments containing Aurora A . 4

5 Kemptide Michaelis -­‐ Menten kemptide A titration using a 2/3 dilution regime was used to concentration, the vary the protocol is detailed below. These assays were performed in buffer containing 50 mM NaCl, 5 , 0.25 mM DTT, 2.5 % glycerol in 10 mM HEPES, pH 7.0 mM MgCl 2 20 μL of buffer was added to 16 wells in one row of a 384 (4.5 -­‐ well plate. 40 μL of kemptide was added to the first well and mixed. 40 μL was transferred from the first well to the mM) th e the 40 μL was second well and mixed. This process was repeated to the 15 well, wher th 4 μM Aurora A ( well left with just buffer. 10 μL of a solution containing ) discarded, and the 16 + [Eu.1] was added to each well and the plate incubated for 15 minutes. 10 μL of a ( 32 μM ), and solution of ATP (4 mM) was added and the plate read immediately. Luminescence intensities – 366 nm and emission using time -­‐ resolved measurements at 292 were taken with excitation at 615 625 nm using an integration time of 60 – 400 μs or intensity at 601 -­‐ 633 nm, at – 75 s). Assays were run in triplicate and the mean of the appropriate time intervals (often For Michaelis -­‐ Menten triplicate intensity values were taken and plotted against time. calculations the initial rate was calculated as the gradient of the intensity against time over the 2 5 % of the enzyme reaction . These initial rates were plotted against kemptide first Menten -­‐ equation concentration and fit to a Michaelis using Origin 2015. Aurora A inhibition /activation assay single point + [Eu.1] kemptide (1 mM), To 20 μL or 0 μM ), and of (16 μM) in 50 mM NaCl, 5 Aurora A (2 μM mM MgCl2, 0.25 mM DTT, 2.5 % glycerol in 10 mM HEPES, pH 7.0 was added 10 μL of the inhibitor /activator (4 μM , 2 μM or 0 μM ) in 10 % DMSO (0% for TPX2 activation) i n a 384 -­‐ well plate mixed and incubated for 15 minutes prior to reading. Immediately before . The wells were r e ading 10 μL of ATP (4 mM) was added to each well . Luminescence intensities were taken with excitation at 292 – 366 nm and emission using time -­‐ resol ved measurements at 615 – 625 nm using an integration time of 60 400 μs . Assays were run in triplicate and the mean of the – triplicate intensity values were taken and plotted against time , and the control with no enzyme intensity taken away. Initial rates were calculated by the gradient of the intensity against time over the first 5 minutes (3 minutes for activation or no inhibitor) of the enzyme reaction. Staurosporine IC 50 conc A titration using a 2/3 dilution regime was used to vary the staurosporine entration, the protocol is detailed below. 5

6 20 μL of 10% DMSO in added to wells 2 to 1 6 of one row of a 384 -­‐ well plate. 60 μL of buffer was st buffer staurosporine ( well. 40 μL was taken from was added to the 1 200 μM), 10% DMSO in nd th st the 1 transferred to the 2 and well and mixed. This was re peated down to the 15 well, , th well left without any staurosporine . 10 μL of where the 40 μL was discarded and the 16 + mM), Aurora A (200 nM) and [Eu.1] kemptide ( (32 μM) was added to each well, the wells 2 tely before reading 10 μL of ATP (4 mM) mixed and the plate incubated for 15 minutes. Immedia Luminescence intensities were taken with excitation at 292 366 nm was added to each well. – resolved measurements at 615 – 625 nm using an integration time of and emission using time -­‐ – 400 μs . Assays were run in t riplicate and the mean of the triplicate intensity values were 60 . Initial rates were calculated by the taken and plotted against time intensity against change in 1 and , plotted against staurosporine concentration time over the first fitted to a 5 minutes oidal curve to calculate the IC . sigm 50 ADP -­‐ Glo assay (staurosporine IC ) 50 A 1 in 2 dilution regime was used to vary the concentration of staurosporine . 20 μL of 10% -­‐ well plate. 40 μL of staurosporine DMSO in buffer was added to wells 2 to 8 of one row of a 384 st st well, (200 μM) in 10% DMSO in buffer was added to the 1 well. 20 μL was taken from the 1 nd th repeated down to the 8 well and mixed. This was , where the 20 μL and transferred to the 2 was discarded. 10 μL of kemptide (2 mM) and Aurora A (200 nM) was added to each well, the wells mixed and the plate incubated for 15 minutes. 10 μL of ATP (4 mM) was added to each well , and the plate incubated for 15 minutes. 15 μL of the enzyme reaction solution was to a 96 Glo reagent in appropriate well Sterilin white plate containing 15 μL of ADP -­‐ transferred -­‐ wells The wells were mixed and the plate incubated for 40 minutes . 30 μL of ADP -­‐ Glo detection . reagent wa s added to the wells, the wells were mixed and the plate incubated for 1 hour. The plate was read by reading the full luminescence emission. Assays were run in triplicate , with a late the duplicate calibration line of varying ATP:ADP ratios, which was used to calcu of ADP in the wells . Initial rates were calculated as the [ADP] formed over the 15 concentration minute enzyme reaction, and the mean of the triplicate values calculated. The initial rates were plotted against staurosporine concentration and fitte d to a sigmoidal curve to calculate the IC . 50 LgtC enzyme assay + UDP -­‐ galactose (2 mM), MgCl (16 μM) and ( 4 mM ), [Eu.1] To 20 μL of a solution containing 2 in 10 mM HEPES, pH 7.0 in a 384 well plate was added 10 μL of a solution -­‐ 0.02% Triton -­‐ X 100 6

7 -­‐ 1 of 2.4, 1.2, 0.6 or 0 U mL ), and the plate incubated for 15 minutes. 10 μL of lactose ( ( 40 LgtC ) was added and the plate read immediately. Luminescence intensities were taken with mM 292 – 366 nm and emission using time -­‐ resolved measurements at 6 15 – 625 nm excitation at – 400 μs, at appropriate time intervals ( 45 s ). Assays were run in using an integration time of 60 triplicate and the mean of the triplicate intensity values were taken and plotted against time. (no enzyme ) experiments were taken away LgtC. from experiments containing Bleaching PDE enzyme assay + and if appropriate, ( 10 mM ) , [Eu.1] To 20 μL of a solution containing MgCl (16 μM) 2 (60 μM) in 10 mM HEPES, pH 7.0 in a 384 -­‐ well plate was added 10 calmodulin (2 μM) and CaCl 2 -­‐ 1 osphodiesterase ( 0.6, 0.3, 0.15 or 0 U mL ), and the plate incubated for 15 ph μL of a solution of ) was added and the plate read immediately. minutes. 10 μL of cAMP or cGMP ( 4 mM Luminescence intensities were taken with excitation at 292 -­‐ and emission using time – 366 nm ed measurements at 615 resolv 625 nm using an integration time of 60 – 400 μs, at – appropriate time intervals. Assays were run in triplicate and the mean of the triplicate intensity values were taken and plotted against time. (no enzyme ) experiments we re taken Bleaching PDE. Initial rates for the calmodulin experiments were away from experiments containing calculated as the gradient of intensity over time for the first 30% of the enzyme reaction. Simultaneous enzyme reaction s + l as ( 10 mM ) , [Eu.1] 20 μL of a solution containing MgC (16 μM) and ATP or ADP ( 2 mM) , 2 appropriate -­‐ well plate. 5 μL of an appropriate , in 10 mM HEPES, pH 7.0, was added to a 384 hexokinase solution and 5 μL of an appropriate pyruvate kinase solution were added. The wells were mixed and incubated for 10 minutes. 10 μL of a solution containing phosphor(enol)pyruvate (4 mM) and glucose (4 mM) was added to all the wells and the plate read immediately. Luminescence intensities were taken with excitation at 292 – 366 nm and emission using time -­‐ resolved measurements at 615 – 625 nm using an integration time of 60 – 400 μs, at appropriate time intervals. Assays were run in triplicate and the mean of the triplicate intensity values were taken and plotted against time. 7

8 Anion screen + mission spectra of [ Eu.1 ] . . Effect of the addition of phosphoanions (1 mM) on the e 1 Figure S + Eu.1 ] = 330 nm , 1 mM phosphoanion , 10 mM HEPES, pH 7.0, λ Conditions: 8 μM [ exc 2. Plate -­‐ based real -­‐ Figure time monitoring of a kinase reaction. a) Kinase simulation in S + , 8 μM [Eu.1] , 10 mM HEPES, pH 7.0), standard assay conditions (1 mM ATP+ADP, 5 mM MgCl 2 measuring the emission intensity (λ = 615 – 625 nm , λ 631 nm) of differing ratios of = 601 -­‐ em exc -­‐ time monitoring of a mo del kinase ATP/ADP (% conversion of ATP to ADP). b) Real + resolved luminescence intensity of [Eu.1] (hexokinase) using the time , conditions: 1 mM ATP, -­‐ + , 10 mM glucose, 8 μM [Eu.1] 5 mM MgCl , 10 mM HEPES, pH 7.0, λ = 292 – 366 nm , λ = 6 01 em 2 exc -­‐ 31 nm . 6 3 . Real -­‐ time monitoring of the hexok inase catalysed phosphorylation of glucose at Figure S + + ] Eu.1 . Conditions: 1 mM ATP, 10 mM glucose, 1 – 16 μM [ different concentrations of [ ] Eu.1 , 5 mM MgCl , 10 mM HEPES, pH 7.0, λ – = 292 – 366 nm , λ nm, integration time 60 = 6 15 -­‐ 625 2 exc em 400 μs, measurements taken every 30 s, average of triplicate reactions 8

9 Different [ATP/ADP] Fluorometer Figure S 4 . -­‐ based kinase simulation at different [anion]: 0.1 mM (a) and b)), 0.5 mM (c) and d)), 1 mM (e) and f)), 2 mM (g) and h)) and 5 mM (i) and j) ATP or ADP. a), c), e), g) + and i) Emission spectra of [ Eu.1 ] on increasing ADP/ATP ratio, b), d), f), h) and j) Plot of + emission intensity ratio 616/599 nm versus percentage conversion of ATP to ADP. 8 μM [ Eu.1 ] , 330 nm variable [ATP+ADP], 5 mM MgCl = , 10 mM HEPES, pH 7.0, λ exc 2 9

10 Figure S Plate -­‐ based kinase simulation using time . resolved method with different anion 5 -­‐ (ATP+ADP) concentrations: 0.1 (a), 0.2 (b), 0.3 (c), 0.5(d), 0.75 (e), 1.0 (f), 1.5 (g), 2.0 (h), 3.0 (i) + ] and 5.0 (j) mM [ATP] + [ADP]. 8 μM [ , variabl e [ATP+ADP], 5 mM MgCl Eu.1 , 10 mM HEPES, 2 pH 7.0, λ = 292 – 366 nm , λ 400 μs = 6 15 -­‐ 625 nm, integration time 60 – em exc Figure S 6 Real -­‐ time monitoring of the hexokinase catalysed phosphorylation of glucose at . + different concentrations of ATP. Conditions: va Eu.1 ] , 5 mM riable ATP, 10 mM glucose, 8 μM [ 1 -­‐ – , 0. U mL MgCl hexokinase, 10 mM HEPES, pH 7.0, λ 625 = 292 2 366 nm , λ = 6 15 -­‐ nm, em exc 2 400 μs, measurements taken every 60 s, average of triplicate reactions integration time 60 – 10

11 ] Different [MgCl 2 . re S 7 ]: 0 mM (a) and b)), 1 mM Fluorometer -­‐ based kinase simulation at different [MgCl Figu 2 (c) and d)), 3 mM (e) and f)), 5 mM (g) and h)) and 10 mM (i) and j) MgCl . a), c), e), g) and i) 2 + Emission spectra of [ ] Eu.1 on increasing ADP/ATP ratio, b), d), f), h ) a nd j) Plot of emission + intensity ratio 616/599 nm versus percentage conversion of ATP to ADP. 8 μM [ Eu.1 ] , 1 mM = 330 nm ATP+ADP, 0 – 10 mM MgCl , 10 mM HEPES, pH 7.0, λ 2 exc 11

12 . Plate -­‐ based kinase simulation using time -­‐ resolved method with differe nt MgCl Figure S 8 2 + concentrations: 0 (a), 1 (b), 1.5 (c), 2 (d), 3 (e), 5 (f), 10 (g), and 20 (h) mM MgCl . 8 μM [ Eu.1 ] , 2 1 mM [ATP+ADP], variable MgCl 625 , 10 mM HEPES, pH 7.0, λ = 292 – 366 nm , λ -­‐ = 6 15 em exc 2 nm, integration time 60 – 400 μs Figure S Real -­‐ tim e monitoring of the hexokinase catalysed phosphorylation of glucose at . 9 + . Conditions: 1 mM ATP, 10 mM glucose, 8 μM [ Eu.1 ] , variable different concentrations of MgCl 2 -­‐ 1 , 0.5 U mL hexokinase, 10 mM HEPES, pH 7.0, λ MgCl nm = 292 – 366 nm, λ 625 = 6 15 -­‐ , em exc 2 400 μs, measurements taken every 60 s, average of triplicate reactions integration time 60 – 12

13 Different [NaCl] Fluorometer Figure S 10 . -­‐ based kinase simulation at different ionic strengths ([NaCl]) : 0 mM (a) and b)), 50 mM (c) and d)), 100 mM (e) and f) ), 200 mM (g) and h)) and 500 mM (i) and j) NaCl. + a), c), e), g) and i) Emission spectra of [ Eu.1 ] on increasing ADP/ATP ratio, b), d), f), h) and j) Plot of emission intensity ratio 616/599 nm versus percentage conversion of ATP to ADP. 8 μM + [ Eu.1 ] = 330 nm , 1 m M ATP+ADP, 5 mM MgCl 500 mM NaCl, 10 mM HEPES, pH 7.0, λ , 0 – 2 exc 13

14 Different buffer concentrations Figure S 11 . Fluorometer -­‐ based kinase simulation at different [Tris]: 10 mM (a) and b)), 20 mM (c) and d)), 50 mM (e) and f)), and 100 mM (g) and h)) Tris, pH 7.5. a), c), e), and g) Emission + spectra of [ Eu.1 ] on increasing ADP/ATP ratio, b), d), f), h) Plot of emission intensity ratio and + [ Eu.1 ] , 1 mM [ATP+ADP], 5 616/599 nm versus percentage conversion of ATP to ADP. 8 μM , 10 mM MgCl = 330 nm – 100 mM Tris, pH 7.5, λ exc 2 14

15 Hexokinase different substrates Figure S . Real -­‐ time monitoring of the hexokinase catalysed phosphorylation of glucose, 12 + ] mannose and fructose. Conditions: 1 mM ATP, 10 mM sugar, 8 μM [ , 5 mM MgCl , 10 mM Eu.1 2 HEPES, pH 7.0 , λ 400 μs, = 292 – 366 nm, λ = 6 15 -­‐ 625 nm, integration time 60 – em exc measurements taken every 60 s, average of triplicate reactions + Concentration of [Eu.1] 13 . Figure S using different resolved method Plate -­‐ based kinase simulation using time -­‐ + ations of [ Eu.1 ] 1 mM [ATP+ADP], 5 mM : 0.5 μM (a), 1 μM concentr (b), 2 μM (c), 4 μM (d ). MgCl 400 – , 10 mM HEPES, pH 7.0, λ nm, integration time 60 = 292 – 366 nm , λ = 6 15 -­‐ 625 em exc 2 μs 15

16 time monitoring of the hexokinase catalysed phosphorylat 14 . Real -­‐ Figure S ion of glucose at -­‐ 1 + three enzyme concentrations (2, 1, 0.5 and 0 U mL using different concentrations of [ Eu.1 ] ) : . Conditions: 1 mM ATP, 10 mM glucose, a) 0.125 , b) 0.25 , c) 0.5 , d) 1, e) 2, f) 4 and g ) 8 μM + ] variable [ Eu.1 nm, , 5 mM MgCl 625 , 10 mM HEPES, pH 7 .0, λ = 292 – 366 nm , λ -­‐ = 6 15 em 2 exc 400 μs, measurements taken every 30 s, average of triplicate reactions integration time 60 – 16

17 Different pH 15 Figure S . Fluorometer -­‐ based kinase simulation at different pHs: pH 7.0 (a) and b)), pH 7.5 (c) + g) Emission spectra of [ and d )), pH 8.0 (e) and f)) and pH 8.5 (g) . a), c), e), and on ] Eu.1 and h) Plot of emission intensity ratio 616/599 nm versus increasing ADP/ATP ratio, b), d), f) + , 50 mM Tris, ] percentage conversion of ATP to ADP. 8 μM [ , 1 mM [ATP+ADP], 5 mM MgCl Eu.1 2 = 330 nm variable pH, λ exc 17

18 Additives . Figure S Fluorometer -­‐ based kinase simulation with different additives: 0.2 mg/mL BSA (a) 16 and b)), 0.02% Triton X -­‐ 100 (c) and d)), and 10% glycerol (e) and f)). a), c), and e) Emission + spectra of [ Eu.1 ] on increasing ADP/ATP ratio, b), d), and f) of emission intensity ratio + 616/599 nm versus percentage conversion of ATP to ADP. 8 μM [ Eu.1 ] , 1 mM [ATP+ADP], 5 = 330 nm mM MgCl , 10 mM HEPES, pH 7.0, λ 2 exc 18

19 resolved method with different buffer . Plate -­‐ based kinase simulation using time -­‐ 17 Figure S 00 mM KCl (c), 10 % DMSO (d), 10 % glycerol (e), 0.01 additives: None (a), 200 mM NaCl (b), 2 -­‐ 1 + and 2 mM DTT (h) . 8 μM [ % Triton BSA (g) , -­‐ , 1 mM [ATP+ADP], 5 Eu.1 ] X 100 (f), 0.1 mg mL 366 nm nm, integration time 60 , 10 mM HEPES , pH 7.0, λ = 292 – mM MgCl , λ 625 = 6 15 -­‐ – em exc 2 400 μs 19

20 . Real -­‐ time monitoring of the hexokinase catalysed phosphorylation of glucose at 18 Figure S -­‐ 1 ) in the presence of various additi ves: a) none, three enzyme concentrations (2, 1 and 0.5 U mL -­‐ 1 b) 200 mM NaCl, c) 10% Glycerol, d) 10% DMSO, e) 2 mM DTT, f) 0.1 mg mL BSA and g) 0.01% + Triton -­‐ X 100. Conditions: 1 mM ATP, 10 mM glucose, 8 μM [ Eu.1 ] , 10 mM HEPES, , 5 mM MgCl 2 = pH 7.0, λ 400 μs, measurements – 292 – 366 nm , λ time 60 = 6 15 -­‐ 625 nm, integration em exc taken every 30 s, average of triplicate reactions 20

21 2+ Different M : MgCl Fluorometer -­‐ based kinase simulation with different MCl 19 . Figure S (a) and b)), and 2 2 + . (c) and d)) CaCl a) and c) Emission spectra of [ Eu.1 ] d) of on incre asing ADP/ATP ratio, b) and 2 + emission intensity ratio 616/599 nm versus percentage conversion of ATP to ADP. 8 μM [ Eu.1 ] , 0 mM HEPES 1 mM [ATP+ADP], 5 mM MCl , 1 = 330 nm , pH 7.0 , λ exc 2 + CaCl Figure S Plate -­‐ based kinase simulation using time -­‐ resolved me thod with . 20 . 8 μM [ Eu.1 ] , 2 1 mM [ATP+ADP], 5 mM = 620 nm, integration Ca Cl , 10 mM HEPES, pH 7.0, λ = 330 nm, λ exc em 2 time 60 – 400 μs 21

22 Temperature Figure S . Plate -­‐ based kinase simulation using time -­‐ resolved method at different 21 + (a), 25 °C (b), 30 °C (c), 35 °C (d), 40 °C (e), and 45 °C (f). 8 μM [ Eu.1 ] , 1 temperatures: 19.6 °C mM [ATP+ADP], 5 mM MgCl nm, , 10 mM HEPES, pH 7.0, λ = 292 – 366 nm, λ 625 = 6 15 -­‐ em exc 2 400 μs integration time 60 – 22 . Real -­‐ time monitoring of the hexokinase catal ysed phosphorylation of glucose at Figure S -­‐ 1 three enzyme concentrations (2, 1, 0.5 and 0 U mL ) at different temperatures: a) 20.5 °C, b) 25 + °C, c) 30 °C, d) 35 °C and, e) 40 °C. Conditions: 1 mM ATP, 10 mM glucose, 8 μM [ Eu.1 ] , 5 mM 400 MgCl , 10 mM HEPES, pH 7.0, λ – = 292 – 366 nm , λ nm, integration time 60 = 6 15 -­‐ 625 em exc 2 μs, measurements taken every 30 s, average of triplicate reactions 22

23 23 Real -­‐ time monitoring of the hexokinase catalysed phosphorylation of fructose at Figure S . against time at different fructose Change in intensity s of fructose. different concentration A) to a Michaelis Menten concentrations, b) Fit of initial rate against fructose concentration -­‐ + -­‐ 1 ] , 5 mM MgCl equation , 0.25 U mL Conditions: 1 mM ATP, 8 μM [ Eu.1 hexokinase, 10 mM 2 15 400 μs, = 292 HEPE 366 nm , λ = 6 S, pH 7.0, λ -­‐ 625 nm, integration time 60 – – em exc measurements taken every 75 s, average of triplicate reactions Figure S 24 . Real -­‐ time monitoring of the a ctivation of AurA by TPX2 (a) , showing a n in crease . initial reactio n rate (b) , shown as a % activity with no TPX2 Conditions: 1 mM ATP, 1 μM AurA, + 0.5 mM kemptide, 0.25 mM DTT, 5 mM MgCl , 2.5% glycerol, 50 mM NaCl, 10 mM , 8 μM [Eu.1] 2 = 615 = – 366 nm HEPES, pH 7.0, λ 292 -­‐ 625 nm, integration time = 60 – 400 μs , λ em exc + 25 . Comparison of staurosporine inhibition IC50 , using [Eu.1] Figure S in real -­‐ time (a) and ADP Glo (b) Conditions: 1 mM ATP, 50 nM AurA, 0.5 mM kemptide, 0.25 mM DTT, 5 mM MgCl , -­‐ 2 + + [Eu.1] [Eu.1] 5% DMSO, 10 mM HEPES, pH 7.0 , for , 2.5% glycerol, 50 mM NaCl, 8 μM : λ = exc 400 μs 292 – 366 nm , λ = 615 -­‐ 625 nm, integration time = 60 – em 23

24 26 . Effect of the addition of various phosphoanions (1 mM) on the e mission spectra of Figure S + + ] . Conditions: [ 8 μM [ Eu.1 ] Eu.1 , 1 mM phosphoanion , 10 mM HEPES, pH 7 .0, λ = 330 nm exc Figure S 27 mission spectra of Effect of the addition of various phosphoanions (1 mM) on the e . + + 8 μM [ ] Eu.1 in the prescence of 5 mM MgCl , 5 mM . Conditions: [ 1 mM phosphoanion ] Eu.1 , 2 MgCl , 10 mM HEPES, pH 7.0, λ = 330 nm 2 exc 24

25 Other enzyme reactions Figure S . Plate -­‐ based enzyme simulations of NTP to NDP enzyme reactions. a) ATP to ADP, b) 28 + Eu.1 ] , 1 mM [NTP + CTP to CDP, c) GTP to GDP, d) UTP to UDP, and e) dATP to dADP. 8 μM [ NDP], 5 mM MgCl nm, integration , 10 mM HEPES, pH 7.0, λ = 292 – 366 nm , λ 625 = 6 15 -­‐ em exc 2 – time 60 400 μs + Pi, b) CTP to 29 Plate -­‐ based NTPase simulations of NTP to NDP + Pi. a) ATP to ADP Figure S . + CDP + Pi, c) GTP to GDP + Pi, d) UTP to UDP + Pi and e) dATP to dADP + Pi. 8 μM [ Eu.1 ] , 1 mM [NTP + (NDP + Pi)], 5 mM MgCl nm, , 10 mM HEPES, pH 7.0, λ = 292 – 366 nm , λ 625 = 6 15 -­‐ em exc 2 400 μs integration time 60 – 25

26 based nucleoside triphosphate pyrophosphohydrolase simulation of NTP to . Plate -­‐ 30 Figure S c) GTP to GMP + PPi and d) UTP to UMP + NMP + PPi. a) ATP to AMP + PPi, b) CTP to CMP + PPi, + Eu.1 ] PPi. 8 μM [ , 1 mM [NTP + (NMP + PPi)], 5 mM MgCl , 10 mM HEPES, pH 7.0, λ – = 292 exc 2 366 nm , λ 400 μs = 6 15 -­‐ 625 nm, integration time 60 – em 31 . Plate -­‐ based enzyme simulations of NTP to NMP + 2 Pi. Figure S a) ATP to AMP + 2 Pi, b) CTP + to CMP + 2 Pi, c) GTP to GMP + 2 Pi and d) UTP to UMP + 2 Pi. 8 μM [ Eu.1 ] , 1 mM [NTP + (NMP + 2 Pi)], 5 mM MgCl nm, integration 625 = 292 – 366 nm , λ = 6 15 -­‐ , 10 mM HEPES, pH 7.0, λ em exc 2 400 μs time 60 – 26

27 Figure S Plate -­‐ b ased enzyme simulations of ATP + NMP to ADP + NDP. a) ATP + AMP to ADP 32 . + NDP, b) ATP + CMP to ADP + CDP, c) ATP + GMP to ADP + GDP and d) ATP + UMP + ADP + + Eu.1 UDP. 8 μM [ , 1 mM [(ATP + NMP) + (ADP + NMP)], 5 mM MgCl , 10 mM HEPES, pH 7.0, λ ] 2 exc 292 – 3 66 nm , λ = = 6 15 -­‐ 625 nm, integration time 60 – 400 μs em -­‐ 33 . Real conversion of phospho(enol) time monitoring of the pyruvate kinase catalysed Figure S pyruvate to pyruvate, using ADP (a), CDP (b), GDP (c), UDP (d) and dADP (e) at three enzyme 1 -­‐ Conditions: 1 mM ) . concentratio ns (2, 1, 0.5 and 0 U mL ND P, 1 mM phosphor(enol) pyruvate , + Eu.1 ] , 5 mM MgCl 8 μM [ , 50 mM KCl, 10 mM HEPES, pH 7.0, λ -­‐ = 292 – 366 nm, λ 15 = 6 em exc 2 0 s, average of tri 625 nm, integration time 60 – 400 μs, measurements taken every 8 plicate reactions 27

28 3 4 . Fluorometer -­‐ based Figure S simulation : UDP -­‐ glucose + glucose to UDP glycosyl transferase + ] a) Emission spectra of [ Eu.1 -­‐ + maltose. on increasing UDP/UDP glucose ratio, b) Plot of tage conversion emission intensity ratio 61 /599 .5 nm versus percen 6.5 UDP -­‐ glucose to UDP . 8 + μM [ Eu.1 ] , , 1 mM [UDP -­‐ glucose + UDP] , 9 mM glucose, 1 mM [glucose + maltose], 2 mM MgCl 2 10 mM HEPES, pH 7.0, λ = 330 nm exc 3 5 . Plate -­‐ based glycosyl transferase simulation (UDP Figure S glucose + glucose to UDP + -­‐ m altose) using time -­‐ resolved method with different UDP -­‐ glucose and UDP concentrations: 0.1 + glucose + UDP. 8 μM [ -­‐ Eu.1 ] (a), 0.2 (b), 0.3 (c), 0.5 (d), 0.75 (e), and 1.0 (f) mM UDP , 10 mM nm [glucose + maltose], 2 mM MgCl , 10 mM HEPES, pH 7.0, λ = 292 – 366 625 , λ = 6 15 -­‐ nm, em 2 exc 400 μs integration time 60 – 28

29 Figure S 6 . Plate -­‐ based glycosyl transferase simulation (UDP -­‐ 3 glucose + glucose to UDP + maltose) using time -­‐ resolved method with different MgCl concentrations: 0 (a), 1 (b), 1.5 (c), 2 2 + nd 5 (f) mM MgCl glucose + UDP], 10 mM [glucose + . 8 μM [ Eu.1 ] -­‐ , 1 mM [UDP (d), 3 (e), a 2 = 292 – 366 nm , λ – maltose], 10 mM HEPES, pH 7.0, λ = 6 15 -­‐ 625 nm, integration time 60 em exc 400 μs -­‐ glucose + glucos Figure S 3 7 . Plate -­‐ based glycosyl transferase simulation (UDP e to UDP + (c), resolved method with different M n Cl maltose) using time concentrations: 10 -­‐ (a), 20 (b), 50 2 + 100 (d) and 200 (e) μ M M n Cl . 8 μM [ Eu.1 ] glucose + UDP], 10 mM [glucose + , 1 mM [UDP -­‐ 2 – maltose], 10 mM HEPES, pH 7.0, λ nm, integration time 60 = 292 – 366 nm , λ = 6 15 -­‐ 625 em exc 400 μs 29

30 Figure S 8 . Fluorometer -­‐ based 3 simulation s a) and b) cAMP to AMP, c) and phosphodiesterase + a) and c) Emission spectra of [ Eu.1 ] on increasing d) cGMP to GMP. AMP/cAMP or GMP/cGMP ratio, b) and d) Plot of emission intensity ratio 61 3 /599 nm versus percentage conversion of + , AMP or GMP . 8 μM [ Eu.1 ] cAMP or cGMP , 1 mM [cAMP + AMP or cGMP + GMP] , 5 mM MgCl to 2 10 mM HEPES, pH 7.0, λ = 330 nm exc Figure S 3 9 . Plate -­‐ based cyclic nucleotide phosphodiesterase simulation (cAMP to AMP) using time -­‐ resolved method with different anion (cAMP+AMP) concentrations: 0.1 (a), 0.2 (b), 0.3 (c), + 0.5 (d), 0.75 (e), and 1.0 (f) mM [cAMP] + [AMP]. 8 μM [ Eu.1 ] , variable [cAMP+AMP], 5 mM MgCl 400 , 10 mM HEPES, pH 7.0, λ – = 292 – 366 nm , λ nm, integration time 60 = 6 15 -­‐ 625 em exc 2 μs 30

31 -­‐ Figure S 40 . Plate based cyclic nucleotide phosphodiesterase simulation (cGMP to GMP) using time -­‐ resolved method with different anion (cGMP+GMP) concentrations: 0.1 (a), 0.2 (b), 0.3 (c), + 0.5 (d), 0.75 (e) , and 1.0 (f) mM [cGMP] + [GMP]. 8 μM [ Eu.1 ] , variable [cGMP+GMP], 5 mM , 10 mM HEPES, pH 7.0, λ 400 MgCl – nm, integration time 60 = 292 – 366 nm , λ = 6 15 -­‐ 625 em 2 exc μs 31

32 P) using 4 1 . Plate -­‐ based cyclic nucleotide phosphodiesterase simulation (cAMP to AM Figure S time -­‐ resolved method with different MgCl concentrations: 0 (a), 1 (b), 1.5 (c), 2 (d), 3 (e), 5 (f), 2 + and 10 (g) mM MgCl . 8 μM [ Eu.1 ] , 1 mM [cAMP+AMP], variable MgCl , 10 mM HEPES, pH 7.0, 2 2 400 μs λ – = 292 – 366 nm , λ = 6 15 -­‐ 625 nm, integration tim e 60 exc em 32

33 Figure S 2 . Plate -­‐ 4 based cyclic nucleotide phosphodiesterase simulation (cGMP to GMP) using -­‐ resolved method with different MgCl time concentrations: 0 (a), 1 (b), 1.5 (c), 2 (d), 3 (e), 5 (f), 2 + and 10 (g) mM MgCl GMP], variable MgCl . 8 μM [ Eu.1 ] , 10 mM HEPES, pH 7.0, , 1 mM [cGMP+ 2 2 366 nm , λ λ = 292 – 400 μs – = 6 15 -­‐ 625 nm, integration time 60 em exc + 10 Figure S 4 3 . (8 μM) in ission spectra of [ Eu.1 ] Effect of various sugars (10 mM) on the em mM HEPES, pH 7.0, λ = 330 nm exc 33

34 time monitoring of the PDE 4 . Real -­‐ 4 -­‐ catalysed conversion of cAMP to AMP , conditions: Figure S + -­‐ , 8 μM [Eu.1] 1 mM cAMP, 5 mM MgCl , 10 mM HEPES, pH 7.0, λ = 615 = 292 – 366 nm , λ em exc 2 400 μs 625 nm, integration time = 60 – S4 5 . Figure Real -­‐ time monitoring of s equential enzyme reactions, involving hexokinase (HK, ATP to ADP) and pyruvate kinase (PK, ADP to ATP). Starting from ADP. Conditions: 1 mM ADP, 1 + mM glucose, 1 mM PEP, 5 mM MgCl , 50 mM KCl, 8 μ M [Eu.1] , 10 mM HEPES, pH 7.0, λ = 292 exc 2 μ s) – 366 nm, λ 400 = 6 15 -­‐ 625 nm, integration time = 60 – em 3 4

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