Analytical methods

3.5.1 Protein concentration determination via UV-absorption spectroscopy

The aromatic amino acids tryptophan, tyrosin and phenylalanine as well as disulfide bonds (cystine) absorb UV-light in a wavelength interval between 250 and 300 nm. The molar extinction coefficient at 280 nm (ɛ280) can be determined from the amino acid composition (Pace et al., 1995) (Equation 8).

!_!"# = !"# ∙ !!"" + !"# ∙ !"#$ + !"#$%&' ∙ !"#

Equation 8: Determination of the molar extinction coefficient ɛ280. ɛ₂₈₀ molar extinction coefficient at 280 nm [M^-1cm^-1]

The specific extinction coefficient (^0.1%A280) can be calculated according to Equation 9 taking the molecular weight into account.

0.1%!_!"# =^!_!"^!"#

Equation 9: Determination of the specific extinction coefficient ^0.1%A₂₈₀.

0.1%A₂₈₀ specific extinction coefficient at 280 nm [cm²/mg]

MW molecular weight of the protein [g/mol]

Using Lambert-Beer’s law, the protein concentration can be determined by measuring the absorbance at 280 nm (Equation 10):

!_!"#= ^0.1%!_!"#∙  !   ∙  !

!   =   !_!"#

!_!"#

!.!% ∙ !    

Equation 10: Determination of the protein concentration by using the specific extinction coefficient ^0.1%A₂₈₀.

A₂₈₀ absorbance at 280 nm c concentration [mg/ml]

d pathlength [cm]

0.1%A₂₈₀ specific extinction coefficient at 280 nm [cm²/mg]

Absorbance spectra between 220 and 350 nm were recorded. The absorbance maximum should be at 278 nm and the A280/A250 ratio should be at least 1.8 for a pure protein solution. No absorbance above 300 nm should be detectable to exclude any distortion of the results caused by light scattering resulting from aggregation.

Table 2 shows the molar extinction coefficients, molecular weights, and specific extinction coefficients of the wild-type proteins used in this work. For protein variants the respective coefficients were recalculated.

Table 2: Properties of wild-type Dr0930 and wild-type PTE relevant for concentration determination.

Number of tryptophan and tyrosine residues, molar extinction coefficients, molecular weight (MW) and specific extinction coefficients of investigated wild-type proteins.

protein Σ Trp + Tyr ɛ₂₈₀ [M^-1cm^-1] MW [g/mol] ^0.1%A₂₈₀ [cm/mg]

wild-type PTE 4 × Trp + 5 × Tyr 29450 36420 0.81

wild-type Dr0930 3 × Trp + 9 × Tyr 29910 34728 0.86

3.5.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Proteins are denatured by the detergent sodium dodecyl sulfate (SDS) and negatively charged in proportion to their molecular weights. SDS binds to the protein in a ratio of approximately one molecule SDS per 1.4 amino acid residues. The net charge of proteins can be neglected compared to the negative charge of the protein complexed with SDS, which yields an approximately uniform mass to charge ratio. As a consequence, electrophoretic mobility depends only on the sieve effect of the gel:

the migration speed is inversely proportional to the logarithm of mass (Laemmli 1970). Table 3 shows the composition of the 12.5% SDS gels used in this work.

Table 3: Composition of a 12.5% SDS-PAGE gel.

Amount specification applies to 13 gels.

resolving gel (12.5%) stacking gel (6%)

resolving/stacking gel buffer 19.5 ml 7.38 ml

acrylamide-SL (30%) 26.2 ml 5.9 ml

H2O 31.58 ml 15.95 ml

TEMED 0.089 ml 0.029 ml

APS (10%) 0.195 ml 0.089 ml

Samples were supplemented 1:4 with 5× SDS-PAGE sample buffer and incubated for 5 min at 95 °C. Gel pockets were loaded with 5-20 µl of sample, and gels were run at 50 mA and 300 V for about 30 min. Subsequently gels were stained with SDS-PAGE staining solution, whereby the detection limit of the Coomassie Brilliant Blue dye G-250 is between 200-500 ng protein/mm². Gels were swayed for 10 min in the staining solution and excess dye was removed by repeatedly boiling in water (microwave 900 W).

Alternatively, ready-to use PhastGel™ Homogeneous-12.5 gels with PhastGel™

Buffer Strips, SDS (GE HEALTHCARE) were used in a PhastSystem™ Separation Control (GE HEALTHCARE) according to the protocols supplied by the manufacturer.

3.5.3 ICP-MS measurements

ICP-MS measurements were performed by Dr. Siddhesh Kamat and Swapnil Ghodge (group of Prof. Dr. F. M. Raushel), using the PerkinElmer DRC II ICP-MS device (software: Eland, PerkinElmer). For this purpose protein buffer was exchanged to metal-free buffer using GE illustra NAP™-25 or PD-10 columns. If applicable, protein and buffer samples were lyophilized for the shipment.

3.5.4 Steady-state enzyme kinetics

3.5.4.1 Colorimetric assay for OPH activity with p-nitrophenol substituted OPs The OPH activity for p-nitrophenyl substituted OPs (compounds 1-7, 4.2.2) was measured in a colorimetric assay (Dumas et al., 1989). The activity was followed by observing the release of the product p-nitrophenol under basic conditions at 400 nm

(ɛ400 = 17000 M^-1 cm^-1) and 30 °C. Reactions were performed in 50 mM CHES, pH 9.0, 100 µM CoCl2 and various concentrations of substrate (Omburo et al., 1992).

For OP compounds 4 and 5 the assay was supplemented with 12% MeOH, due to limited solubility of the substrates. Measurements were performed with a Cary 100 Bio (VARIAN) or V650 (JASCO) spectrophotometer (total volume = 1 ml) or a SpectraMax-340 plate reader (MOLECULAR DEVICES, total volume = 250 µl), using plastic cuvettes and VIS 96-well plates, respectively. The reaction was started by adding the enzyme. Background of free p-nitrophenol, associated with absorbance at 400 nm, as well as limited solubility of substrates restricted the maximum substrate concentration applicable. The concentration of OP compounds 1-7 was determined by titration with KOH prior to steady-state enzyme kinetics (2.10.2).

3.5.4.2 Coupled DTNB (Ellman’s Reagent) assay

The OPH activity for the hydrolysis of phosphorothiolates (OP compound 8, 4.2.2) was measured in a coupled colorimetric assay (adapted from Yang et al., 2003).

Cleavage of the phosphorothiolate bond was followed by inclusion of DTNB (Ellman’s reagent, 5,5’-dithiobis-(2-nitrobenzoic acid)), which reacts with the free thiol released as product. The disulfide bond of DTNB is cleaved yielding 2-nitro-5-thiobenzoate (NTB^-), which ionizes to NTB^2- dianion (yellow color) under basic conditions. The reaction is followed at 412 nm (ɛ412 = 14150 M^-1 cm^-1) and 30 °C. Reactions were performed in 50 mM HEPES pH 8.0, 0.3 mM DTNB, 100 µM CoCl2, 12% MeOH, and various concentrations of substrate. The concentration of OP compound 8 was determined enzymatically using wild-type PTE (2.10.2).

3.5.4.3 pH-dependent colorimetric assay

The hydrolysis of δ-nonanoic lactone (δ-NL; 4.2.2) was monitored using a pH-sensitive colorimetric assay (Chapman & Wong, 2002; Khersonsky & Tawfik, 2005).

Proton release from carboxylic acid formation was followed using the pH-indicator m-cresol purple. The reactions were performed in 2.5 mM BICINE (initial pH: 8.3), 0.1 mM CoCl2, 1.4% DMSO, 0.1 mM m-cresol purple, 0.2 M NaCl and various concentrations of substrate (0-2 mM). The change in absorbance at 577 nm was monitored at 30 °C.

The proteins Dr0930 and PTE were stored in 20 mM HEPES pH 7.5, 100 µM CoCl2 or 50 mM HEPES pH 8.5, 100 µM CoCl2, respectively. Prior to use, the buffer was exchanged to 10 mM bicine pH 8.3, 100 µM CoCl2 using a NAP-25 desalting column. The assay mixture was pre-incubated for 10 min at 30 °C to obtain a constant baseline, and the reaction was started by adding the enzyme. The conversion factor was determined by in-situ calibration with acetic acid (change of absorbance A577 per change in proton concentration, ɛ577 = 1.17 × 10³ M^-1cm^-1). A slight background rate was observed in the absence of any enzyme, presumably due to acidification of the reaction assay by atmospheric CO2 (Khersonsky & Tawfik, 2005). The background rate was independent of the substrate concentration, and subtracted from the initial rates.

3.5.4.4 Data analysis

Steady-state kinetic parameters were determined by fitting the experimental data of the saturation curves to the Michaelis-Menten equation (Equation 11), using the computer program SigmaPlot.

!   =  !_!"#∙ [!]

!_!+ [!]

Equation 11: Michaelis-Menten Equation.

v: initial velocity v_max: maximum velocity S: substrate concentration KM: Michaelis constant

The catalytic rates (v) were corrected for background rate of spontaneous hydrolysis in the absence of enzyme. If the substrate concentration required to saturate an enzyme variant was higher than the maximal solubility of the substrate, Vmax/KM was determined by a linear regression of the initial part of the saturation curve.

3.5.5 Dixon plot for competitive inhibition

The Dixon plot is used to determine the type of enzyme inhibition and the dissociation constant (Ki) for an enzyme-inhibitor complex (Segel, 1993). The velocity equation for competitive inhibition can be converted to a linear form in which the varied ligand is [I] (Equation 12).

!

!   =   !_!

!_!"#∙ [!] ∙ !_!  [!] +   !

!_!"#   ! +  !_! [!]  

Equation 12: Velocity equation for competitive inhibition in linear form.

v: initial velocity v_max: maximum velocity S: substrate concentration KM: Michaelis constant K_i: dissociation constant

The effect on velocity (v) is determined over a range of inhibitor concentrations [I].

A plot of 1/v versus [I] at a constant unsaturating concentration of [S] (here: [S] = KM) yields a straight line with a positive slope. If the inhibition is known to be competitive, Ki can be determined from the slope of a linear fit of the data points according to Equation 12. The type of enzyme inhibition is confirmed to be competitive, when the linear graph intersects with the y-achses (1/v) at 1/vmax and the x-achses ([I]) at -Ki.

3.5.6 Determination of stereopreference

The enzymatic hydrolysis of racemic OP compounds was monitored as a function of time. The change of A400 over time is an exponential time courses. Depending on the compound and enzyme variant used, one or two phases can be observed and fit by a single- or double exponential fit according to Equation 13.

! ! = !_!+ !   ! − !^!!!!

! ! = !_!+ !   ! − !^!!!! +  !   ! − !^!!!!

Equation 13: Single- and double-exponential fit of an exponential time course.

A(t): absorption at time t y0: off-set (in absorbance)

a, b: magnitudes of exponential phases k₁, k₂: observed rate constants

t time

If a double-exponential fit did not converge due to limited data points, either the observed rate constant of the second phase, k2, or the magnitude of the second exponential phase, b, were constrained. The substrate concentration was kept below the KM value; hence, the time course follows pseudo-first order kinetics and the associated rate constants are directly proportional to the amount of enzyme added (Cleland, 1970; Nowlan et al., 2006). The catalytic efficiency, kcat/KM, was estimated from the exponential components according to Equation 14.

!_!"#

!_! = !_! [!]

Equation 14: Determination of the catalytic efficiency of individual enantiomers.

k_x: observed rate constant [E]: enzyme concentration

To identify the configuration of the non-hydrolyzed isomer in solution after the reaction has approached an end point at approximately half amplitude, wild-type PTE or engineered PTE variants, with previous reported stereopreference for 4-acetylphenol leaving group analogues of OPs 1-5 (Tsai et al., 2010a), were added to the reaction mixture, inducing the (exclusive) hydrolysis of the residual isomer.