P R Ortiz de Montellano

The Akt kinase signals directly to endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries.

The role of cytochrome P-450 in the myogenic response of isolated, perfused renal arcuate arteries of dogs to elevations in transmural pressure was examined. The phospholipase A2 inhibitor oleyloxyethylphosphorylcholine (1 and 10 microM) inhibited the greater than threefold increase in active wall tension in these arteries after an elevation in perfusion pressure from 80 to 160 mm Hg. Inhibition of cyclooxygenase activity with indomethacin (1 or 10 microM) had no effect on this response. The cytochrome P-450 inhibitors ketoconazole (10 and 100 microM) and beta-diethyl-aminoethyldiphenylpropylacetate (SKF 525A, 10 and 100 microM) also inhibited the myogenic response. At a pressure of 160 mm Hg, SKF 525A (10 microM) and ketoconazole (100 microM) reduced active wall tension in renal arteries by approximately 70%. Partial inhibition of the myogenic response was obtained after perfusion of the vessels with mechanism-based inhibitors of P-450, 1-aminobenzotriazole (75 microM) and 12-hydroxy-16-heptadecynoic acid (20 microM). The thromboxane receptor antagonist SQ 29,548 (1 or 10 microM) had no effect on the pressure-induced increase in active wall tension in renal arteries. Arachidonic acid (50 microM) constricted isolated perfused renal arteries and potentiated the myogenic response in the presence of indomethacin. This response was completely reversed by ketoconazole (100 microM) or SKF 525A (100 microM). Microsomes (1 mg/ml) prepared from small renal arteries (200-500 microns) and incubated with [1-14C]arachidonic acid (0.5 mu Ci, 50 microM) produced a metabolite that coeluted with 20-hydroxyeicosatetraenoic acid (20-HETE) during reversed-phase high-performance liquid chromatography. The formation of this product was inhibited by both ketoconazole and SKF 525A at concentrations of 10 and 100 microM. These results are consistent with the involvement of the vasoconstrictor 20-HETE and other cytochrome P-450 metabolites of endogenous fatty acids in the myogenic response.

Structure of a non-peptide inhibitor complexed with HIV-1 protease. Developing a cycle of structure-based drug design.

A stable, non-peptide inhibitor of the protease from type 1 human immunodeficiency virus has been developed, and the stereochemistry of binding defined through crystallographic three-dimensional structure determination. The initial compound, haloperidol, was discovered through computational screening of the Cambridge Structural Database using a shape complementarity algorithm. The subsequent modification is a non-peptidic lateral lead, which belongs to a family of compounds with well characterized pharmacological properties. This thioketal derivative of haloperidol and a halide counterion are bound within the enzyme active site in a mode distinct from the observed for peptide-based inhibitors. A variant of the protease cocrystallized with this inhibitor shows binding in the manner predicted during the initial computer-based search. The structures provide the context for subsequent synthetic modifications of the inhibitor.

Mechanism-based probes of the topology and function of fatty acid hydroxylases.

The use of three mechanism‐based probes to investigate the topology and function of fatty acid hydroxylases is discussed. 1) The observation of protein rather than heme alkylation in the reaction of cytochrome P4504A1 with 10‐undecynoic acid supports the argument that the enzyme circumvents the inherent preference for ω‐1 hydroxylation by restricting access to the ferryl oxygen. 2) The regiochemistry of the ferricyanide‐mediated iron‐to‐nitrogen shift of the cytochrome P450102 (P450BM‐3) phenyl‐iron complex indicates that the active site of this bacterial fatty acid hydroxylase is open primarily above pyrrole ring A of the prosthetic heme group, 3) Inhibition of clofibrate‐mediated peroxisome proliferation in cultured rat hepatocytes by inactivation of cytochrome P4504A1 indicates that ω‐hydroxylation of fatty acids provides a signal for peroxisome proliferation.—Ortiz de Montellano, P. R.; Chan, W. K.; Tuck, S. F.; Kaikaus, R. M.; Bass, N. M.; Peterson, J. A. Mechanism‐based probes of the topology and function of fatty acid hydroxylases. FASEB J. 6: 695‐699; 1992.

Arylhydrazines as probes of hemoprotein structure and function

Abstract The reactions of arylhydrazines (ArNHNH2) or aryldiazenes (ArNNH) with simple iron porphyrins or with hemoproteins that have relatively open active sites, including hemoglobin, myoglobin, cytochrome P450, chloroperoxidase, catalase, prostaglandin synthase, and indoleamine-2,3-dioxygenase yield σ-bonded aryl-iron complexes. Denaturation of the protein complexes under aerobic, acidic conditions shifts the aryl group to the porphyrin nitrogens and produces mixtures of the four possible N-arylprotoporphyrin IX regioisomers. The regioisomers are obtained in approximately equal amounts if the iron-to-nitrogen shift occurs outside of the protein but the ratio of isomers differs if the rearrangement is controlled by the protein. Only in the case of cytochrome P450 enzymes can the shift be induced to occur without denaturation of the protein. The isomer ratios obtained when the shift occurs in the intact active site provide direct experimental information on the active site topology and dynamics. Topological information has thus been obtained for cytochromes P450 1A1, 1A2, 2B1, 2B2, 2B4, 2B10, 2B11, 2E1, 11A1, 51, 101, 102, and 108. In contrast to hemoproteins with open active sites, conventional peroxidases react with arylhydrazines to give δ-meso-aryl adducts and covalent protein adducts. Reaction with the δ-meso edge but not the heme iron provides key evidence that restricting access of substrates to the ferryl oxygen helps direct the reaction towards peroxidase rather than peroxygenase catalysis.

Porphyrinogenic activity and ferrochelatase-inhibitory activity of sydnones in chick embryo liver cells

3‐[2‐(2,4,6‐Trimethylphenyl)thioethyl]‐4‐methylsydnone was shown to be a potent porphyrinogenic agent in chick embryo liver cells. The accumulation of protoporphyrin IX was consistent with the finding that ferrochelatase activity was inhibited. 3‐Benzyl‐4‐phenylsydnone did not inhibit ferrochelatase activity and protoporphyrin IX was found to constitute only a minor fraction of the porphyrins. These results support the idea that the porphyrinogenicity of 3‐[2‐(2,4,6‐trimethylphenyl)thioethyl]‐4‐methylsydnone is due to its catalytic activation by cytochrome P‐450 leading to heme alkylation and formation of N‐vinylprotoporphyrin IX which inhibits ferrochelatase.

Crystal Structure and Properties of CYP231A2 from the Thermoacidophilic Archaeon Picrophilus torridus

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.

Protein dynamics and imidazole binding in cytochrome P450 enzymes

P450 (cytochrome P450) enzymes have major roles in the biosynthesis of endogenous factors such as steroids and eicosanoids, in the termination of the action of endogenous factors such as retinoic acid, in the metabolism of most drugs and xenobiotics and in the generation of toxic and carcinogenic products. Understanding the determinants of the substrate and inhibitor specificities of these enzymes is important for drug design. The crystallographic analysis of the deformability of two bacterial P450 active sites associated with the binding of azole (a class of inhibitors with an imidazole or triazole ring that co-ordinates to the haem iron) inhibitors described in the present study illustrates the importance of protein conformational malleability in the binding of imidazole derivatives.

Protein control of the formation and decomposition of the CYP119 and CYP101 aryl-iron complexes.

Abstract CYP119, the first thermophilic P450 enzyme, reacts much more slowly than CYP101 (P450cam) with aryldiazenes to give σ-bonded aryl-iron complexes. The CYP119 complexes are stable anaerobically at 80 °C but are readily oxidized by O2 to give the N-arylprotoporphyrin IX regioisomers. The aryl shift can also be initiated in the absence of O2 by K3Fe(CN)6. In contrast, the corresponding CYP101 complexes are insensitive to O2 but decompose at temperatures above 50 °C owing to denaturation of the protein. The rate of the CYP119 aryl shift is decreased by electron-withdrawing substituents, with ρ=−1.50 for both the O2- and K3Fe(CN)6-dependent reactions. A similar dependence (ρ=−0.90) is observed for the K3Fe(CN)6-dependent CYP101 shift. The enthalpies and entropies of activation suggest that the CYP119 and CYP101 K3Fe(CN)6-mediated reactions are similar, but the CYP119 O2-dependent reaction proceeds via a different transition state. In all cases, the rate-determining step is oxidation of the aryl-iron complex. The temperature dependence of the O2- and K3Fe(CN)6-dependent CYP119 shifts provides evidence for temperature-dependent equilibration of two active site conformations. The oxygen sensitivity of the CYP119 aryl-iron complexes, and the temperature dependence of their rearragement, reflect the unique active site properties of this thermophilic P450 enzyme.

4.09 – Peroxidases*

The human peroxidases are hemoproteins that utilize H2O2 to oxidize a variety of endogenous and exogenous substrates. The well-established members of this enzyme family are eosinophil peroxidase, lactoperoxidase, myeloperoxidase, and thyroid peroxidase. The reactions catalyzed by these enzymes, which include halide oxidation and substrate free radical formation, contribute to cellular pathology and xenobiotic toxicity. In contrast to the peroxidases, the peroxiredoxins are cysteine-containing proteins that detoxify both H2O2 and alkyl peroxides at the expense of electron donors such as thioredoxin and cellular thiols. They do not oxidize other endogenous or xenobiotic substrates. The peroxiredoxins appear to be constituents of oxidative stress signaling pathways and help to ameliorate oxidative stress, but do not appear to contribute significantly to xenobiotic toxicity.