Binding to the Prosthetic Heme

P450 is often irreversibly inactivated via covalent attachment of the catalytically activated inhibitor, or a fragment of it, to the heme group. A heme alkylation mechanism has been unambiguously demonstrated, in many instances, by evidence of equivalent activity and heme loss and the isolation and structural characterization of the modified hemes. It must be noted that in the absence of explicit evidence for heme adduct formation, an equimolar loss of enzyme content and heme does not unambiguously establish that heme alkylation is responsible for enzyme inactivation because alternative mechanisms exist for the catalysis-dependent destruction of the heme (see Section 3.4). It is also possible for a heme adduct 

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C. Compounds That Covalently Bind to the Prosthetic Heme... 

Figure 7.18. The oxidation of phenelzine (PhCHjCHjNHNHj) and phenylhydrazine (PhNHNHj) by P450 produces carbon radical products that bind to the prosthetic heme group. In the case of phenyihydrazine, the phenyl radical binds first to the iron atom to give a complex that subsequently rearranges under oxidative conditions to the iV-phenyl adduct. It is not known if phenelzine initially forms a similar but less stable carbon-iron intermediate.

Covalent Binding to the Prosthetic Heme and Modification of the P450 Protein by Heme Fragments... 

Figure 9.1 CYP catalytic cycle. The sequential two-electron reduction of CYP and the various transient intermediates were first described in the late 1960s [206], The sequence of events that make up the CYP catalytic cycle is shown. The simplified CYP cycle begins with heme iron in the ferric state. In step (i), the substrate (R—H) binds to the enzyme, somewhere nearthe distal region of the heme group and disrupts the water lattice within the enzymes active site [207], The loss of water elicits a change in the heme iron spin state (from low spin to high spin) [208]. Step (ii) involves the transfers of an electron from NADPH via the accessory flavoprotein NADPH-CYP reductase, with the electron flow going from the reductase prosthetic group FAD to FMN to the CYP enzyme [206,209]. The...

Chloramphenicol and secobarbital exhibit properties similar to those of tienilic acid, but they have not been studied in humans (11). Oxidative dechlorination of chloramphenicol with formation of reactive acyl chlorides appears to be an important metabolic pathway for irreversible inhibition of CYP. Chloramphenicol binds to CYP, and subsequent substrate hydroxylation and product release are not impaired. The inhibition of CYP oxidation and the inhibition of endogenous NADPH oxidase activity suggest that some modification of the CYP has taken place, which inhibits its ability to accept electrons from the CYP reductase (11). Secobarbital completely inactivates rat CYP2B1 functionally, with partial loss of the heme chromophore. Isolation of the N-alkylated secobarbital heme adduct and the modified CYP2B1 protein revealed that the metabolite partitioned between heme N-alkylation, CYP2B1 protein modification, and epoxidation. A small fraction of the prosthetic heme modifies the protein and contributes to the CYP2B1 inactivation (12). 

Certain CYPs undergo mechanism-based inactivation as a result of conversion of their prosthetic heme groups to products that irreversibly bind to the protein. Hydrogen peroxide and cumene hydroperoxide partially degrade the prosthetic heme to monopyrrole and dipyrrole fragments that bind to the protein (24). Presently, no drugs have been shown to fall into this class. 

Another class of cytochrome P-450 inhibitors, compounds with a monosubstituted acetylenic function, are well known for their potential as insecticide synergists (21) and some have already been reported to be active as JH biosynthesis inhibitors as well (19, 22). Ortiz de Montellano and Kunze (23) have shown that many ethynyl substrates cause the destruction of rat hepatic cytochrome P-450, when the prosthetic heme is alkylated during attempted metabolism of the triple bond. Such suicide substrates must bind to the enzyme and be catalytically acceptable thereby offering a potential for selectivity. In fact, selectivity of suicide substrates for particular molecular forms (isozymes) of hepatic... 

Mechanism-based irreversible inhibition occurs when a reactive metabolic intermediate is formed in situ that can (1) bind covalently with the prosthetic heme through N-alkyllary-lation (e.g., secobarbital), f"2 alkylate the apo-cytochrome (e.g., chloramphenicol or 2-ethy-nylnaphthalene), or (5) cause destruction of the prosthetic heme to products that irreversibly bind to the apocytochrome (e.g. CCI4) (158). These mechanism-based inactivators have primarily been designed and used for the selective inhibition of specific CYP enzymes and elucidating the mechanism of P450 reactions. Some drugs (e.g., aromatase inhibitors)... 

Similar studies with f/-ans-4-hydroxy-2-none-nal (HNE, a cytotoxic byproduct of biological membrane lipid peroxidation), indicate that it is also metabolically activated by CYP2B1 and -2B4 to a reactive species that binds irreversibly to their prosthetic heme"". Unlike the mechanism-based inactivation by aromatic aldehydes, strucmral analyses of the corresponding heme adduct (MW 770) revealed that the reaction proceeds without deformylation and involves an acyl carbon radical that partitions between addition to the heme and formation of the carboxylic acid"". Together these findings suggest that the P450-mediated metabolic activation of aldehydes is a versatile process wherein the enzyme may be inactivated via mechanistically diverse heme modifications. 

The NOSs are best characterized as cytochrome P-450-like hemeprot-eins (Bredt et al., 1991 Stuehr and Ikeda, 1992 White and Marietta, 1992). They can be broadly divided into a reductase domain at the COOH terminus and an oxidative domain at the NH2 terminus (Fig. 1). The primary amino acid sequences of NOS isoforms share common consensus sequence binding sites for calmodulin, NADPH, flavin-adenine dinucleotide (FAD), and flavin mononucleotide (FMN) (Bredt et al., 1991 Marsden et al., 1992 Sessa et al., 1992 Xie et al., 1992 Lyons et al., 1992 Lowenstein et al., 1992). Each enzyme functions as a dimeric protein in catalyzing the NADPH-dependent five-electron oxidation of L-arginine to generate NO. L-Citrulline is a by-product (Back et al., 1993 Abu and Stuehr, 1993). Electrons are supplied by NADPH, transferred along the flavins and calmodulin, and presented to the catalytic heme center (Stuehr and Ikeda, 1992 White and Marietta, 1992). The NOS apoenzyme requires tetrahydrobiopterin, prosthetic heme (ferroprotoporphyrin IX), calmodulin, FMN, and FAD as cofactors for monomer assembly and/or catalytic activity (Abu and Stuehr, 1993 Mayer and Werner, 1994 Kwon etal., 1989 Stuehr and Ikeda, 1992 Stuehr and Griffith, 1992 White and Marietta, 1992 McMillan etal., 1992 Klatt... 

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