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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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Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD).Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 20 nM.
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pone-0010601-g003: Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD).Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 20 nM.

Mentions: For all reactions studied, increasing the final peroxidative substrates' concentration (at an initial constant peroxide concentration) generally gave an increase in the reaction rates for the sub-millimolar reaction range studied (Figure 2 is a salient example for ABTS, other data are shown in Table 1), which could be fit to non-linear regression of Michaelis-Menten equation, with R2 values above 0.9. However, the data could not be transformed to fit the popular linearized models exemplified by Lineweaver-Burk or Eadie-Hofstee (results not shown). Also, incrementing the first substrate concentration by decades did not give corresponding levels of increase in second substrate conversion, as expected in Figure 1 (left side). The reaction profiles obtained by varying peroxide concentrations at constant peroxidative substrate concentration gave mixed trends (Figure 3 & Table 1 show the profile for TMPD). The increase in peroxide concentrations gave an apparent inhibition at higher concentrations, the extent of which varied with different peroxidative substrates. Again, none of these profiles could be fit to a non-linear substrate inhibition model within an extension of the Michaelis-Menten paradigm (as exemplified by the Belanger fit [15]) to give global and reproducible constants with accuracy and precision (Table 1).


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)

Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD).Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 20 nM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010601-g003: Kinetics of CPO catalyzed peroxidation of TMPD obtained by varying the peroxide concentration, at constant peroxidative substrate (TMPD).Initial conditions- pH 3.5, 100 mM phosphate buffer, 30°C, [CPO] = 20 nM.
Mentions: For all reactions studied, increasing the final peroxidative substrates' concentration (at an initial constant peroxide concentration) generally gave an increase in the reaction rates for the sub-millimolar reaction range studied (Figure 2 is a salient example for ABTS, other data are shown in Table 1), which could be fit to non-linear regression of Michaelis-Menten equation, with R2 values above 0.9. However, the data could not be transformed to fit the popular linearized models exemplified by Lineweaver-Burk or Eadie-Hofstee (results not shown). Also, incrementing the first substrate concentration by decades did not give corresponding levels of increase in second substrate conversion, as expected in Figure 1 (left side). The reaction profiles obtained by varying peroxide concentrations at constant peroxidative substrate concentration gave mixed trends (Figure 3 & Table 1 show the profile for TMPD). The increase in peroxide concentrations gave an apparent inhibition at higher concentrations, the extent of which varied with different peroxidative substrates. Again, none of these profiles could be fit to a non-linear substrate inhibition model within an extension of the Michaelis-Menten paradigm (as exemplified by the Belanger fit [15]) to give global and reproducible constants with accuracy and precision (Table 1).

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