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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
Reconstituted CYP2C9 system shows depletion of specific hydroxylated product over time.Initial concentrations are ∼0.08 uM of CYP2C9  =  CPR, 0.04 to 0.8 uM Cytb5, 1 mM NADPH. Other details are mentioned in the experimental section.
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pone-0010601-g010: Reconstituted CYP2C9 system shows depletion of specific hydroxylated product over time.Initial concentrations are ∼0.08 uM of CYP2C9  =  CPR, 0.04 to 0.8 uM Cytb5, 1 mM NADPH. Other details are mentioned in the experimental section.

Mentions: Figure 9 shows the 4′hydroxylated product obtained for diclofenac's reaction with two concentrations of CYP2C9. The CYP concentration was shown to have pronounced effect on the value of constants (quite like the results with CYP2E1) and the “substrate inhibition” was more pronounced at higher enzyme concentration. When a similar reaction setup was probed with respect to product formation at various time intervals (Figure 10), it was noted that the formed product disappeared for higher substrate concentration. Incorporation of Cytb5 lowered the product formation at low substrate concentrations but showed a positive effect on the yield of product at higher substrate concentrations.


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)

Reconstituted CYP2C9 system shows depletion of specific hydroxylated product over time.Initial concentrations are ∼0.08 uM of CYP2C9  =  CPR, 0.04 to 0.8 uM Cytb5, 1 mM NADPH. Other details are mentioned in the experimental section.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010601-g010: Reconstituted CYP2C9 system shows depletion of specific hydroxylated product over time.Initial concentrations are ∼0.08 uM of CYP2C9  =  CPR, 0.04 to 0.8 uM Cytb5, 1 mM NADPH. Other details are mentioned in the experimental section.
Mentions: Figure 9 shows the 4′hydroxylated product obtained for diclofenac's reaction with two concentrations of CYP2C9. The CYP concentration was shown to have pronounced effect on the value of constants (quite like the results with CYP2E1) and the “substrate inhibition” was more pronounced at higher enzyme concentration. When a similar reaction setup was probed with respect to product formation at various time intervals (Figure 10), it was noted that the formed product disappeared for higher substrate concentration. Incorporation of Cytb5 lowered the product formation at low substrate concentrations but showed a positive effect on the yield of product at higher substrate concentrations.

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