Encora

Download Encora 1.2

ENCORA 1.2

Encora is a program for enzymatic kinetic parameter fitting using progress curve analysis. It has been developed by R.J.W. Slats, J.W. Hennipman, B. Romein and A.J.J. Straathof at the Delft University of Technology.
The present version, which still has some restrictions, is freely downloadable.
The user is guided through the fitting procedure, and example problem files illustrate its use. The help- and readme files contain detailed information on this.

What does Encora tell you?
If you are studying an enzymatic reaction and assume some of its basic mechanistic features, such as the sequence in which the substrates enter the active site, Encora will choose the rate equation that corresponds to that mechanism. Then it will optimize the values of the kinetic parameters in this rate equation (Km, kcat, Ki, etc.) in order to get the best fit of the equation to your set of progress curves of the enzymatic reaction. Visually you can judge if the fit is acceptable. Off-line you can compare the fit for different mechanisms.

The difference between progress curve analysis and other enzymatic kinetic methods
There are two types of enzymatic kinetic methods:

  • transient (or pre-steady state) methods, which are of special interest if detailed mechanistic information about an enzymatic reaction is required
  • steady state methods, which are easier, but may lead to less detailed information.

Steady-state reactions can be analyzed in two manners: initial rate analysis, where only the first part of a conversion curve is used, and progress curve analysis, where the full curve is used. In initial rate analysis, traditional methods such as Lineweaver-Burk plots can be applied. These can be easy computerized, e.g. in a spreadsheet program, and dedicated software is available as well. Progress curve analysis is more difficult, always requiring dedicated software.

When to use Encora?
When one is interested in only a limited subset of the kinetic properties, e.g. in many mechanistic studies, initial rate analysis is preferred, because it is easier. However, progress curve analysis becomes attractive if one requires the rate equation including the parameters for reaction equilibrium, enzyme inactivation, inhibition, and spontaneous decomposition of substrates and products.

Encora performs progress curve analysis using a set of full conversion curves that is obtained at a fixed pH, temperature and solvent composition. (If only the initial part of these curves would be used, much information about the reaction rate would be lost.) It becomes particularly attractive to use Encora if it is relatively easy to generate the full conversion curve once the experiment has started, for example when the conversion is monitored on-line in a PC-linked pH-stat or spectrometer. In typical cases progress curve analysis performs well with 30-100 data points per curve, without much scatter. The more parameters are to be fitted, the more curves at different initial concentration are needed.

A self-consistent set of parameters can be obtained, but when the kinetic problem is underestimated one obtains no parameter at all. Then the initial part of the curves still may be used to get some of the desired parameters by initial rate analysis.

Not all progress curves can be used
Encora assumes that only one enzyme is active. Isoenzymes must be avoided. Competing reactions catalyzed by the same enzyme or by different enzymes, cannot be dealt with by the present version of the program. This means that one reaction should be studied at a time, and parallel or sequential reaction must be avoided.

Therefore do not use Encora for studying:

  • polymerization or depolymerization reactions (because the reaction products will be converted further by the enzyme)
  • kinetically controlled synthesis reactions (because parallel hydrolysis reactions occur)
  • kinetic resolution of racemates (unless only one enantiomer is converted, or unless R- and S-enantiomers are studied separately)

However, Encora can deal with enzymes that perform an asymmetric synthesis from a prochiral substrate, even when the enantioselectivity is such that the competing reaction to the undesired enantiomer occurs.

Encora assumes that the reaction is carried out in a homogeneous solution. The presence of other phases or interfaces cannot be accounted for. To study the intrinsic kinetics of immobilized enzyme, one should take care that diffusion is much faster than reaction and one should use so little enzyme that the amount of substrate and product in the immobilization matrix is negligible.

Installation
Encora runs on WIN95/98/NT. The installation requires the file ENCORA.ZIP. Install Encora on drive C: (not on other drives) by unzipping it using WINZIP. Encora requires about 2M disk space when fully installed.

How to start?
Do not directly start to work on your actual problem. First take a look at the readme-file. Then take a look at some of the data files in the Examples, using a simple text editor (Wordpad, e.g.); you cannot see the tables of data that are used by the program within the program! After starting the program, cancel the initial window, and take a look at the Help contents. Then try to run one of the exercises. The first real obstacle will be the preparation of your own data file in the correct format.

Background information
Some background on fitting kinetic parameters and the use of Encora for this, including some hints, is given in ref. [1]. The preprint version of this paper can be downloaded. The copyright belongs to Elsevier Science Inc.

In this paper, an example of a parameter fit procedure is worked out. The set of progress curves that was used for this fit procedure has been included as Example 3 in Encora.

The application of Encora for studying the kinetics of a biotransformation catalyzed by R-hydroxynitrile lyase is described in ref. [2].

  1. A.J.J. Straathof. Development of a computer program for analysis of enzyme kinetics by progress curve analysis, Journal of Molecular Catalysis B: Enzymatic, 11 (2001) 991-998.
  2. W.F. Willeman, U. Hanefeld, A.J.J. Straathof, and J.J. Heijnen, Estimation of kinetic parameters by progress curve analysis for the synthesis of (R)-mandelonitrile by Prunus amygdalus hydroxynitrile lyase. Enzyme Microb. Technol., 27 (2000) 423-433.
Training
Encora is used in the annual Advanced Course on Biocatalysis of the Delft University of Technology.

 

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