Limits...
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
Kinetics of CPO catalyzed peroxidation of Pyrogallol (and ABTS in the inset) obtained by varying ABTS concentration to supramillimolar levels, at constant peroxide.Initial conditions- pH 3, 100 mM phosphate buffer, 25°C, [CPO] = 2 nM, peroxide  = 2 mM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2871781&req=5

pone-0010601-g004: Kinetics of CPO catalyzed peroxidation of Pyrogallol (and ABTS in the inset) obtained by varying ABTS concentration to supramillimolar levels, at constant peroxide.Initial conditions- pH 3, 100 mM phosphate buffer, 25°C, [CPO] = 2 nM, peroxide  = 2 mM.

Mentions: Figure 4 shows that a substrate like pyrogallol, which showed lower inhibitions with peroxide increments, also exhibited lowered product (purpurogallin) formation at supra-millimolar concentrations of pyrogallol. The inhibition was also seen for an active site excluded substrate like ABTS (inset of Figure 4). When the two sets of data (rates obtained by varying peroxide at constant final peroxidative substrate versus rates obtained by varying final peroxidative substrate at constant peroxide) were compared, it could be seen that increase in peroxide (at constant peroxidative substrate) had a greater effect on lowering rate. In most cases, the Belanger fit curves did not justifiably cover the data points at higher substrate concentrations (data not shown).


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 Pyrogallol (and ABTS in the inset) obtained by varying ABTS concentration to supramillimolar levels, at constant peroxide.Initial conditions- pH 3, 100 mM phosphate buffer, 25°C, [CPO] = 2 nM, peroxide  = 2 mM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010601-g004: Kinetics of CPO catalyzed peroxidation of Pyrogallol (and ABTS in the inset) obtained by varying ABTS concentration to supramillimolar levels, at constant peroxide.Initial conditions- pH 3, 100 mM phosphate buffer, 25°C, [CPO] = 2 nM, peroxide  = 2 mM.
Mentions: Figure 4 shows that a substrate like pyrogallol, which showed lower inhibitions with peroxide increments, also exhibited lowered product (purpurogallin) formation at supra-millimolar concentrations of pyrogallol. The inhibition was also seen for an active site excluded substrate like ABTS (inset of Figure 4). When the two sets of data (rates obtained by varying peroxide at constant final peroxidative substrate versus rates obtained by varying final peroxidative substrate at constant peroxide) were compared, it could be seen that increase in peroxide (at constant peroxidative substrate) had a greater effect on lowering rate. In most cases, the Belanger fit curves did not justifiably cover the data points at higher substrate concentrations (data not shown).

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