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2.9.1 Continuous ADP Release Assay

This assay is based on the production of ADP via ATP hydrolysis linked to NADH

oxidation, by the coupling enzymes: pyruvate kinase and lactate dehydrogenase (PK/LDH) as described by Wampler and Westhead (1968). If active, VanS proteins should hydrolyse ATP to ADP, and pyruvate kinase will phosphorylate ADP by the catalytic conversion of phospho-enolpyruvate (PEP) to pyruvate, thus regenerating ATP. Lactate dehydrogenase uses pyruvate and converts it to lactate, by oxidising NADH to NAD+ (see Figure 2.9.1.1).

Figure 2.9.1.1:Reaction pathway involved in ADP production by ATP hydrolysis, as described by Wampler and Westhead (1968). VanS or ATP can be used to initiate the reaction, and ATP is catalytically regenerated by pyruvate kinase. The reaction is followed at 340 nm on a UV spectrophotometer, by monitoring oxidation of NADH to NAD+.

A reaction mixture typically consisted of 50 mM HEPES pH 7.6, 10 mM MgCl2, 50 mM

KCl, 2.5 mM phosphor-enolpyruvate (PEP), 0.3 mM NADH (freshly prepared daily), 1 μl of pyruvate kinase/lactate dehydrogenase (PK/LDH, Sigma), 3 mM ATP, and 20 μL to 80 μL of purified VanS (at 2-8 mg/ml or 10 nmol final concentration) in 0.03-0.07% w/v detergent, all in a final volume of 200 μL. Assays were performed on a Cary 100 spectrophotometer.

VanS

VanS -PO

ATP

ADP

Pyruvate Kinase

PEP

Pyruvate

Lactate

Dehydrogenase

Lactate

NADH

NAD

A

[96]

All components except ATP were added to a 1 cm path-length UV-visible quartz cuvette (Hellma), mixed and placed in the cell changer of the spectrophotometer with the temperature controller set at 20oC for all experiments. The cuvette was allowed to

equilibrate to 20oC before setting the absorbance at 340 nm to zero. Sample absorbance was followed for 1 – 5 minutes, until a flat baseline, after which the cuvette was quickly

removed, ATP substrate added and mixed and placed immediately back into the cell changer. The reaction was followed for at least 60 minutes, up to 300 minutes, and the resulting absorbance changes over the time-course of the assay were recorded using Varian Kinetics software. To demonstrate the dependence of the absorbance decrease on both VanS and ATP components, assays were also initiated by addition of VanS.

2.9.2 Continuous Inorganic Phosphate Release Assay

This assay is based on the utilisation of any free inorganic phosphate by the enzyme MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside) and its subsequent conversion by PNP enzyme (purine nucleoside phosphorylase) to methylthioguanine and ribose-1-phosphate, effecting an increase in absorbance, which may be observed at a wavelength of 360 nm, as detailed by Webb (1992). The MESG enzyme has a strong absorbance at 330 nm, whereas methylthioguanine has an absorbance maximum at 360 nm, therefore a change in absorbance occurs which can be followed spectrophotometrically (see Figure 2.9.2.1).

This assay was conducted as a second measure of protein activity, to complement the ADP production assay. It also enabled quantification of the amount of phosphate released by VanS after autophosphorylation and therefore allowed calculation of how much ATP is turned over from this, giving relative stoichiometries, corrected for any free phosphate release.

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Figure 2.9.2.1: Reaction pathway involved in inorganic phosphate release by ATP hydrolysis, as described by Webb (1992). VanS or ATP can be used to initiate the reaction, but ATP is not regenerated. The reaction is followed at 360 nm on a UV spectrophotometer, by monitoring conversion of MESG to methylthioguanine by PNP enzyme.

All components except ATP were added to the quartz cuvette (Hellma), mixed and placed in the cell changer of the spectrophotometer with the temperature controller set at 20oC for all experiments. The cuvette was allowed to equilibrate to 20oC before setting the absorbance at 360 nm to zero. Sample absorbance was followed for 1 – 5 minutes, until a flat baseline, after which the cuvette was quickly removed, ATP substrate added and mixed and placed immediately back into the cell changer. The reaction was followed for 20-60 minutes and the resulting absorbance changes over the time-course of the assay were recorded using Varian Kinetics software. To demonstrate the dependence of the absorbance decrease on both VanS and ATP components, assays were also initiated with VanS enzyme.

A reaction mixture typically consisted of 50 mM HEPES pH 7.6, 10 mM MgCl2, 0.2 mM

MESG, 1 μL of PNP (160 units/mL), 3 mM ATP, and 20 μL up to a limit of 80 μL of purified VanS enzyme (at 2-8 mg/ml or 10 nmol final concentration) in 0.03-0.07% w/v detergent, all in a final volume of 200 μL. Assays were performed on a Cary 100 spectrophotometer (Varian). In both assays, careful pipetting was required to reduce air bubbles or bubbles in detergent containing samples.

VanS

VanS -PO

ATP

ADP

PNP

MESG + Pi

Methylthioguanine

+ Ribose-1-Phosphate

A

[98]

2.9.3 Identification of Assay products by Anion Exchange Chromatography

To identify products of the reaction between VanS and ATP, simplified experiments were conducted involving only VanS enzyme and ATP substrate (i.e. ATP is not regenerated). A reaction mix consisted of 50 mM HEPES pH 7.6, 1 mM MgCl2, 1 mM KCl, 0.03% DDM

detergent, 10 nmol of VanS enzyme purified in 0.03% DDM and 3 mM ATP in a final volume of 200 μL. The reaction time was monitored, from the point at which ATP was added, and all 200 μL samples (aliquoted from a master mix) were maintained at 20oC in a PCR machine for direct comparison with activity data. Control samples were also run and tested simultaneously. These control samples were in-house standards of AMP, ADP or ATP (at 3 mM final concentration) in a reaction mix containing 50 mM HEPES pH 7.6, 1 mM MgCl2 with a final volume of 200 μL. A reaction mix was also tested involving VanS and

ADP; 50 mM HEPES pH 7.6, 1 mM MgCl2, 1 mM KCl, 0.03% DDM detergent, 10 nmol of

VanS enzyme purified in 0.03% DDM and 3 mM ADP in a final volume of 200 μL.

For all samples, a 200 μL aliquot was removed from the PCR machine at specific time points (0, 30, 60, 120, 240, 360 or 480 min) and the reaction was quenched with 25 mM (final concentration) of EDTA pH 8, which chelates to any magnesium ions present. The sample was then applied to a new 5 kDa MWCO concentrator, and concentrated for 20 minutes at 4000 g, to obtain the VanS protein in the upper chamber and the DNA in the lower chamber. nucleotide products of the reaction could then be analysed by Anion Exchange

Chromatography (IEX), and identified against standards (AMP/ADP/ATP). 80 μL of the flow through from the concentrator was diluted with 1.3 mL of buffer A (10 mM ammonium acetate pH 7.6), and applied via a 1 mL injection loop onto a pre-equilibrated MonoQ 5/50 GL column (Amersham) on an AKTA purifier at 1 mL/min flow rate. Absorbance at 280 and 254 nm was recorded, and ~10 mL was passed through the column to elute any unbound compounds, before a gradient was started of 100% B (1M ammonium acetate pH 7.6), over 30 minutes, and held at 100% B for 10-15 minutes.

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Once all compounds had been eluted, the gradient was lowered to 0% B and the next sample was loaded onto the column when the conductivity reached 0 mS/cm. Absorbance traces were recorded for each sample, and the integrals of all peaks and their retention time (relative to standards) enabled percentages of AMP/ADP/ATP to be calculated.