Explaining the Atypical Reaction Profiles of Heme Enzymes with a Novel Mechanistic Hypothesis and Kinetic Treatment

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.

Silica Sol-Gel Entrapment of the Enzyme Chloroperoxidase

“…To determine optimal reaction conditions, we varied the pH value of the assay buffer and the concentrations of pyrogallol and hydrogen peroxide. Our observations for free CPO agree well with previous studies [24,29]. Acidic conditions, with pH values in the range of 3.5 to 4.5, and pyrogallol concentrations at approximately 35 mM resulted in optimum catalytic performance.…”

“…The poor quality of the fit shown in Figure 1(b) for the data on sol-gel entrapped CPO might be caused by large data point variations and the limitations of the substrate inhibition model. Manoj et al [29] demonstrated that the substrate inhibition model used here (see (1)) can yield acceptable fits for individual substrate variations, but global fit parameters were shown to be unattainable [29]. For example, the and values of one substrate depended on the concentration of the other cosubstrate.…”

Identification of the Active Components in Shenmai Injection that Differentially Affect Cyp3a4-Mediated 1′-Hydroxylation and 4-Hydroxylation of Midazolam

“…Recent advances have demonstrated that CYP3A4 cooperativity involves the binding of multiple (at least 2) substrate molecules to the binding pocket and represents a case of true allostery characterized by effector-induced conformational transitions (Davydov and Halpert, 2008). On the basis of the above theory and our results, the differential effects of PXT on the metabolism of MDZ, similar to the effectors of citral, 6-gingerol, and D-limonene (Zhang and Lim, 2008), can be explained by a two-site model (Korzekwa et al, 1998;Domanski et al, 2001) or a multi-site model (Kenworthy et al, 2001;Manoj et al, 2010;Smirnov et al, 2011) of CYP3A4. It can be hypothesized that PXT can bind to one defined site that is also occupied by MDZ in the position required for 4-hydroxylation (or 19-hydroxylation) and that both substrates can displace each other.…”

Dioxygenases Catalyze O-Demethylation and O,O-Demethylenation with Widespread Roles in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

“…However, substrate inhibition of protopine 6-hydroxylase resulting from the elevated cellular pool of protopine or related alkaloids could potentially result in the decreased accumulation of sanguinarine. Substrate inhibition has been reported for salutaridine reductase involved in morphine biosynthesis in opium poppy (35) and is common in mammalian CYPs (36,37).…”