Enzyme titration reaction scheme.

<p>E is the <i>Torpedo californica</i> acetylcholinesterase enzyme, I the m-(N,N,N-trimethylammonio) trifluoroacetophenone (TMTFA) inhibitor, and EI their complex; <i>k₀</i> is a second order association rate constant. The reaction was drawn using the <i>Reaction Scheme</i> tab of ENZO.</p

Cholinesterase reaction with a substrate.

<p>The reaction scheme and the corresponding differential equations were created by ENZO. Substrate (butyrylthiocholine) bound to the peripheral anionic site is denoted by “S” on the left of the label name (e.g., SE, SES, SEA, SEAS). When bound to the catalytic anionic site, the “S” is placed on the right of the name (e.g., ES, SES, EAS SEAS). Covalent acyl-enzyme is represented by EA and P is the first product (thiocholine) released upon enzyme acylation. The acyl group is denoted by A.</p

Converged results of parameter fitting for enzyme active site titration experiment.

<p>Initial concentrations of enzyme E and inhibitor I for progress curve files tfk1.dat, tfk2.dat, tfk3.dat (<i>Experimental Data</i> panel shows tfk1.dat) are fitted in the interval of [0, 10²⁰]; the initial concentration of EI is zero and fixed; the checkbox “E” is checked under <i>Measured Species</i>, which signifies that E is the measured quantity and the progress curves below represent the time course of its residual activity. The respective units of the residual activity in the Y-axis are OD/min and the units of time in X-axis are seconds. Fitted rate constant <i>k₀</i> and initial values of E and I at three different concentrations of I are displayed under the <i>Evaluated Parameters</i> in the upper right corner panel, the experimental progress curves are blue and the fitted curves are red as shown in <i>Time Course of the Reaction</i> chart at the bottom panel of the screen. The arrows mark the difference between the inital value and the plateau.</p

Normalization curve for the determination of active site concentration from enzyme activity.

<p>X-axis represents the concentration of added TMTFA corresponding to actual enzyme concentrations after 300 times delution. Y-axis represents the difference between the initial enzyme activity and the activity at plateau caused by the presence of experimental TMTFA concentration. The concentration of the substrate in all activity determinations was 0.5 mM.</p

Michaelis-Menten reaction scheme.

<p>E is the free enzyme, S the substrate, ES the Michaelis complex and P the product; <i>k₀</i> is a second order and <i>k₁</i> and <i>k₂</i> are first order rate constants, respectively. The differential equations were automatically generated from the drawn reaction scheme by ENZO.</p

Autoactivation of procathepsin B.

<p>The data originally presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022265#pone.0022265-Rozman1" target="_blank">[11]</a> were used. The total amount of protein was determined by Pace et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022265#pone.0022265-Pace1" target="_blank">[13]</a> and used as a fixed value for each substrate/precursor concentration. The initial values of ES is zero and E is fitted in the interval [0, 1]. The initial value of rate constant <i>k₀</i> is set to diffusion rate value of 10⁸ M⁻¹ min⁻¹ and fixed, while initial values of <i>k₁</i> and <i>k₂</i> are 200 min⁻¹ and 7.2 min⁻¹ respectively, and fitted in the interval of [0, 10²⁰]. The sum of free active enzyme (E) and the instantaneusly dissociated complex (ES) is the measured species. Y-axis represents the concentration of cathepsin B in molar concentration and the X-axis represents time in minutes. The final estimated values of rate constants and initial concentrations of active enzyme portion for each individual curve are displayed under the <i>Evaluated Parameters</i>.</p

ENZO URL:

<p><a href="http://enzo.cmm.ki.si" target="_blank">http://enzo.cmm.ki.si</a><b>.</b> The ENZO web page provides a short introduction and links to a quick guide, examples and ENZO tool.</p