applications > All applications
Enzyme Kinetic Measurements for a Combinatorial Library of Inhibitors of Pseudomonas Elastase

George Cathcart, Brendan Gilmore, Brian Walker, School of Pharmacy, QUB, Belfast, 10/2009


  • Pseudomonas elastase activity monitored using Abz-Nba internally quenched substrate
  • Km and Vmax calculated using MARS Data Analysis Software
  • Michaelis Menten kinetics determined for a library of 160 elastase inhibitors
Click here for a PDF version of this application note.

Request more information


Pseudomonas elastase (pseudolysin, LasB) is a metalloprotease virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa.(1) As one of the main virulence factors of this bacterium, it contributes to chronic and intractable infection in various disease states from the cystic fibrosis lung, to chronic ulcers of the skin.

The central role of LasB makes it a key drug target in this process, and so a library of inhibitor candidates was developed for screening against this enzyme.(2) Assays were performed using the FLUOstar OPTIMA microplate reader, which allowed highly adaptable data capture, and screening of multiple compounds in parallel. (Figure 1) Data was analysed directly within the MARS software, which allowed extraction of subsets of data post-assay.

Fig. 1: BMG LABTECH FLUOstar OPTIMA multidetection plate reader.

Assay Principle

The assay principle is shown in figure 2.

Fig. 2: Assay principle for the determination of LasB activity.

The internally quenched protease substrate Abz-peptide-Nba (2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide) gives only a low fluorescence signal. After cleavage of the peptide bound by LasB the fluorescent donor group cannot transfer the energy to the quenching acceptor group resulting in a high fluorescence signal which is directly related to the enzymatic activity.

Materials and Methods

  • Abz-Ala-Gly-Leu-Ala-Nba (Peptides International, US)
  • FLUOstar OPTIMA, BMG LABTECH, and MARS Data Analysis Software
  • Library of LasB inhibitors, synthesised at The School of Pharmacy, Queens’ University, Belfast.

Kinetic measurement

LasB was prepared at 1 in 1000 dilution from 100 µg/mL stock, and used at 10 µL per well, giving a working concentration of 1 ng of LasB per well.
The Km of the substrate was first calculated by assay of a series of concentrations of substrate from 20 µM to 1000 µM, against a fixed concentration of LasB.

Inhibitor studies

Stock solutions of inhibitors were prepared in DMF at 10 mM, and further diluted when required. Instrument settings employed were as follows:

No. of flashes per well: 10
Target temperature: 37°C
Excitation filter: 330/10 nm
Emission filter: 460/10 nm

All assays were performed in buffer containing 0.05 M TRIS HCL, 2.5 mM CaCl2, 1 % DMF, pH 7.2, across a range of concentrations of inhibitor.

Results and Discussion

The results can be seen in figure 3, followed by graphical display of the rate of hydrolysis vs substrate concentration (figure 4), and a double reciprocal or Lineweaver-Burk plot, figure 5.

Fig. 3: Km determination for the LasB substrate Abz-Ala-Gly-Leu-Ala-Nba.

Fig. 4: Rate of substrate hydrolysis by LasB vs substrate concentration.

Fig. 5: Km determined by Linewaever-Burk plot. The double reciprocal of the data from figure 4 is used to linearise the data.

The slope of the line on the Lineweaver-Burk plot gives Km / Vmax, while the X- intercept gives - 1 / Km, and the Y- intercept, 1 / Vmax. The data from figure 5 can therefore be used to calculate Km by solving the equation of the line Y = mX + c, where m = slope.

Fig. 6: Progress Curves for hydrolysis of substrate by LasB in the presence of a range of concentrations of a typical LasB inhibitor. Data taken from reference 2.

Linear transformation provides a value for the slope of the line, according to the equation y = mx + c. The Ki could be determined for each inhibitor in turn, via the Michael Menten equation (figure 7 and table 1).

Fig. 7: Linear transformation of progress curves for a typical LasB inhibitor.


Table 1: Ki Values (µM) for inhibitor library. ‘NI’ (No Inhibition) has been stated for values over 1000 µM. Values in grey identify a general trend for low Ki values in inhibitors containing P’1 Trp and Tyr residues. (Data was taken from reference 2.)


The FLUOstar OPTIMA offers convenient calculation of Km, adaptable assay optimization, parallel assay of multiple inhibitors, and isolation of subsets of data post-assay.


  1. Liu, P.V. (1974) J. Infect. Dis. 130, S 94-99.
  2. Cathcart, G.R., Gilmore, B.F., Walker, B. (2009) Bioorg. Med. Chem. Lett. 19, (21).