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Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment.

Manoj KM, Baburaj A, Ephraim B, Pappachan F, Maviliparambathu PP, Vijayan UK, Narayanan SV, Periasamy K, George EA, Mathew LT - PLoS ONE (2010)

Bottom Line: 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.With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth.The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

View Article: PubMed Central - PubMed

Affiliation: Center for BioMedical Research, Vellore Institute of Technology University, Vellore, Tamilnadu, India. muralimanoj@vit.ac.in

ABSTRACT
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.

Show MeSH
Steady state kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP with NADPH added directly, as a function of incorporated Cytb5.Experimental details are given in methods section.
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pone-0010601-g005: Steady state kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP with NADPH added directly, as a function of incorporated Cytb5.Experimental details are given in methods section.

Mentions: Figures 5 & 6 and Table 2 depict the result when substrate effect was probed in two reaction systems- (1) employing NADPH regeneration system with enzymes to remove reduced oxygen species (ROS) and (2) directly with NADPH and without enzymes included to remove ROS. In both these reaction systems, employment of moderate amounts of Cytb5 enhanced the product formation at lower substrate concentrations when compared to the controls. Notably, the first setup gave much higher yields of product in comparison to the second. However, incorporation of excess Cytb5 gave similar product formations (especially at higher substrate concentrations ranges) for both setups. The effect of CYP concentration and reproducibility was probed and the results are shown in Figure 7 & Table 2. Clearly, the magnitude of KIS varied from one experiment to another significantly. The reaction profile was charted in detail by taking a 16 point curve (Figure 8), which showed that the descending part of the Belanger fit (after inflexion) significantly deviated from the experimental points. A theoretical simulation using the double hyperbolic combination showed a similar profile as the experimentally determined points (Figure 8).


Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment.

Manoj KM, Baburaj A, Ephraim B, Pappachan F, Maviliparambathu PP, Vijayan UK, Narayanan SV, Periasamy K, George EA, Mathew LT - PLoS ONE (2010)

Steady state kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP with NADPH added directly, as a function of incorporated Cytb5.Experimental details are given in methods section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871781&req=5

pone-0010601-g005: Steady state kinetics of reconstituted CYP2E1 enzymatic system mediated conversion of pNP with NADPH added directly, as a function of incorporated Cytb5.Experimental details are given in methods section.
Mentions: Figures 5 & 6 and Table 2 depict the result when substrate effect was probed in two reaction systems- (1) employing NADPH regeneration system with enzymes to remove reduced oxygen species (ROS) and (2) directly with NADPH and without enzymes included to remove ROS. In both these reaction systems, employment of moderate amounts of Cytb5 enhanced the product formation at lower substrate concentrations when compared to the controls. Notably, the first setup gave much higher yields of product in comparison to the second. However, incorporation of excess Cytb5 gave similar product formations (especially at higher substrate concentrations ranges) for both setups. The effect of CYP concentration and reproducibility was probed and the results are shown in Figure 7 & Table 2. Clearly, the magnitude of KIS varied from one experiment to another significantly. The reaction profile was charted in detail by taking a 16 point curve (Figure 8), which showed that the descending part of the Belanger fit (after inflexion) significantly deviated from the experimental points. A theoretical simulation using the double hyperbolic combination showed a similar profile as the experimentally determined points (Figure 8).

Bottom Line: 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.With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth.The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

View Article: PubMed Central - PubMed

Affiliation: Center for BioMedical Research, Vellore Institute of Technology University, Vellore, Tamilnadu, India. muralimanoj@vit.ac.in

ABSTRACT
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.

Show MeSH