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.
Highlights
Hemoproteins serve multiple roles in the cellular biochemistry and as a result, they are one of the well-studied proteins
CYP is a typical monooxygenase which requires a ternary mixture of molecular oxygen, yet another enzyme called cytochrome P450 reductase (CPR) and redox equivalents from NADPH [9]
The reaction profiles obtained by varying peroxide concentrations at constant peroxidative substrate concentration gave mixed trends (Figure 3 & Table 1 show the profile for tetramethyl phenylene diamine (TMPD))
Summary
Hemoproteins serve multiple roles in the cellular biochemistry and as a result, they are one of the well-studied proteins. 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.
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