12 research outputs found
Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment
Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the “steady state kinetics” of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of “the product of interest” in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of “substrate concentration at maximum observed rate”. The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes
CYP2E1 kinetics, probed in detail using experimental data and theoretical simulation.
<p>Experimental details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010601#s4" target="_blank">methods</a> section.</p
The newly proposed mechanistic possibilities in reactions catalyzed by hemoproteins are shown.
<p>At the left are two-electron (top) and one-electron (bottom) oxidized enzyme intermediates, giving rise to two-electron (top) and one-electron (lower) oxidized product or intermediates in the center. The final products that could be formed are shown to the right.</p
The new biphasic fit is plotted for CYP2E1 mediated hydroxylation profile for conversion of pNP from <b>Figure 8</b>.
<p>The new biphasic fit is plotted for CYP2E1 mediated hydroxylation profile for conversion of pNP from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010601#pone-0010601-g008" target="_blank"><b>Figure 8</b></a>.</p
Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD).
<p>Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 20 nM.</p
Reconstituted CYP2C9 system shows depletion of specific hydroxylated product over time.
<p>Initial concentrations are ∼0.08 uM of CYP2C9 = CPR, 0.04 to 0.8 uM Cyt<i>b</i><sub>5</sub>, 1 mM NADPH. Other details are mentioned in the experimental section.</p
Analyses of CYP catalyzed peroxidations by Belanger fits.
<p>Kinetic constants shown in the first three columns are average of duplicate experiments (with standard deviation) and 95% confidence intervals respectively.</p
Analyses of CPO catalyzed peroxidations by Belanger fits.
<p>Kinetic constants shown in the first three columns are average of duplicate experiments (with standard deviation) and 95% confidence intervals respectively.</p><p>*pH 3, CPO = 1.8 nM, 25°C.</p
Steady state kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP with NADPH added directly, as a function of incorporated Cytb<sub>5</sub>.
<p>Experimental details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010601#s4" target="_blank">methods</a> section.</p
State kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP, checked for reproducibility and precision.
<p>Experimental details are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010601#s4" target="_blank">methods</a> section.</p