Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H(2)O(2) compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H(2)O(2) and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H(2)O(2) and HCN while features that inhibit substrate ionization slow the reactions.
"CYP2J2-Mediated Metabolism of Endocannabinoids each substrate tested, rapid mixing and formation of the heme-cyanide complex red-shifted the Soret (Fig. 6A). As shown in Fig. 6B, the difference spectra D (A 444–414nm ) were analyzed as a function of time and yielded an exponential biphasic curve that was comprised of a fast and slow phase (Bidwai et al., 2013). The fast phase corresponds to the rate of cyanide binding to the ferric heme active site. "
[Show abstract][Hide abstract] ABSTRACT: The endocannabinoids anandamide (AEA) and 2 -arachidonoylglycerol (2-AG) are arachidonic acid derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well-known for the metabolism of arachidonic acid (AA) to the biologically active epoxyeicosatrienoic acids (EETs). Herein we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids (EET-EA) whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides (EET-G). Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH dependent reaction with CYP2J2 and cytochrome P450 reductase (CPR). The formation of the predominant AEA and 2-AG epoxides were confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small molecule ligand egress and molecular modeling. The experimental and theoretical data were in agreement which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane bound cardiovascular epoxygenase.
Journal of Pharmacology and Experimental Therapeutics 10/2014; 351(3). DOI:10.1124/jpet.114.216598 · 3.97 Impact Factor
"They were constructed by simultaneously replacing Arg48, Trp51, and His52 with either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). We have previously reported on the reaction of these mutants with hydrogen peroxide and cyanide . As anticipated, the reaction with H2O2 is substantially reduced but the peroxygenase activity is increased by a factor of 34. "
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the "peroxide shunt" pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific "peroxygenation" catalysts.
The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig's blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25[degree sign]C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044 +/- 0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150 +/- 0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction.
Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.
[Show abstract][Hide abstract] ABSTRACT: CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2) into thromboxane A2 (TXA2), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.
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