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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.

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The newly proposed mechanistic possibilities in reactions catalyzed by hemoproteins are shown.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.
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pone-0010601-g012: The newly proposed mechanistic possibilities in reactions catalyzed by hemoproteins are shown.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.

Mentions: The kinetic profiles obtained in this work indicate that the single electron abstractions (which have been proven to not necessarily be finally catalyzed at a unique catalytic site by CPO [10]) show a lowered yield of product upon increasing any one of its substrates. The data from this work indicates that lowering of product yields at high substrate concentrations may not be owing to a process involving interaction of the substrate and the enzyme alone, for both CPO and CYP. Evidence presented in the reactions with CYPs show that these observations could very well be owing to other factors, as is shown in Figure 12. The mechanistic route could involve:


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)

The newly proposed mechanistic possibilities in reactions catalyzed by hemoproteins are shown.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010601-g012: The newly proposed mechanistic possibilities in reactions catalyzed by hemoproteins are shown.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.
Mentions: The kinetic profiles obtained in this work indicate that the single electron abstractions (which have been proven to not necessarily be finally catalyzed at a unique catalytic site by CPO [10]) show a lowered yield of product upon increasing any one of its substrates. The data from this work indicates that lowering of product yields at high substrate concentrations may not be owing to a process involving interaction of the substrate and the enzyme alone, for both CPO and CYP. Evidence presented in the reactions with CYPs show that these observations could very well be owing to other factors, as is shown in Figure 12. The mechanistic route could involve:

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