In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min ؊1 ) with relatively low K m (24 M); nevertheless, at concentrations of >100 M, the rate of pNP oxidation gradually decreased as a second molecule bound to CYP2E1 through an effector site (K ss ‫؍‬ 260 M), which inhibited activity at the catalytic site. 4-Methylpyrazole (4MP) was a potent inhibitor for both sites through a mixed inhibition mechanism. The K i for the catalytic site was 2.0 M. Although we were unable to discriminate whether an EIS or ESI complex formed, the respective inhibition constants were far lower than K ss . Bicyclic indazole (IND) inhibited catalysis through a single CYP2E1 site (K i ‫؍‬ 0.12 M). Similarly, 4MP and IND yielded type II binding spectra that reflected the association of either two 4MP or one IND molecule(s) to CYP2E1, respectively. Based on computational docking studies with a homology model for CYP2E1, the two sites for monocyclic molecules, pNP and 4MP, exist within a narrow channel connecting the active site to the surface of the enzyme. Because of the presence of the heme iron, one site supports catalysis, whereas the other more distal effector site binds molecules that can influence the binding orientation and egress of molecules for the catalytic site. Although IND did not bind these sites simultaneously, the presence of IND at the catalytic site blocked binding at the effector site.CYP2E1 (P450 or CYP for a particular isoform) is a mammalian cytochrome P450 enzyme, which oxidizes a structurally diverse class of endogenous and exogenous (xenobiotic) compounds (1, 2). A majority of studies have focused on the role of CYP2E1 in phase I metabolism of xenobiotic compounds, e.g. drugs, food additives, and environmental contaminants. Growing evidence also supports an important physiological role for CYP2E1 in gluconeogenesis. CYP2E1 is regulated similarly to enzymes contributing to gluconeogenesis in relation to starvation and diabetes and, in fact, recognizes precursors to gluconeogenesis, acetone, acetol (1-hydroxyacetone), and fatty acids (3) as substrates.Nevertheless, the selectivity that governs the transformation of molecules by CYP2E1 is poorly understood. A better knowledge of the molecular features that confer specificity of substrates allows predictions to be made as to pharmaco-and toxico-kinetic properties, and such insights are ultimately exploitable in novel drug development and assessment of risk associated with exposure to environmental chemicals. Moreover, the catalytic capacity for CYP2E1 makes the enzyme an excellent target for engineering specific catalytic properties for commercial production of specialty chemicals and remediation activities into plants and other organisms, as reported recently (4, 5).CYP2E1 has broad substrate specificity toward typically small (molecular weight Ͻ 100) and hydrophobic molecules (2, 6). Of the more than 70...

Synthesis of various cyclic carbonates with yield up to 100% and turn over frequency (TOF) of 351 h -1 using CO 2 and epoxides and a cobalt (III) complex of tetraamidomacrocyclic ligand is described. The catalyst was characterized by single crystal X-ray crystallography. A study of reaction conditions indicates that 2 MPa pressure of CO 2 without any co-solvent is sufficient to achieve the desired product.

The "Experimental Procedures" and "Results" sections contained errors in nomenclature and in references to specific models and schemes, respectively. PAGE 3489:The following sentence, "Similar to the traditional mechanism for competitive inhibition, the inhibitor could bind only to free enzyme at the catalytic site to yield single-site inhibition," should include a reference to model 1 of Scheme 2.The following sentence, "Both substrate and inhibitor could alter binding of the other molecule (model 1)," should reference model 2a of Scheme 2 instead of model 1.The following sentence, "For model 2, only substrate acted allosterically, such that substrate affected inhibitor binding (K i K si ) but inhibitor did not affect substrate binding (K s ϭ K is )," should reference model 2b of Scheme 2 instead of model 2.The following sentence, "Model 3 described the alternative possibility wherein inhibitor was the only allosteric effector," should reference model 2c of Scheme 2 instead of model 3.The following sentence, "In the absence of allosterism (traditional noncompetitive inhibition, model 4), all inhibition constants were the same and the ESI and EIS complexes were equivalent," should reference model 2d of Scheme 2 instead of model 4. PAGE 3490:The following sentence, "CYP2E1 demonstrated a relatively low K ss (24 M) and the rapid turnover of 47 min Ϫ1 for pNP; nevertheless, at higher pNP concentrations (Ͼ100 M), the activity gradually decreased as a second molecule bound to CYP2E1 through an effector site (K s 260 M), which inhibited activity at the catalytic site (Table 3)," was incorrect. It should read as follows. "CYP2E1 demonstrated a relatively low K m (24 M) and the rapid turnover of 47 min Ϫ1 for pNP; nevertheless, at higher pNP concentrations (Ͼ100 M), the activity gradually decreased as a second molecule bound to CYP2E1 through an effector site (K ss 260

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentrationdependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photolabeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364 and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.

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We report a heterogeneous cobalt−phosphinebased water oxidation catalyst that was produced by thermal synthesis, and can be easily and rapidly deposited onto a variety of substrates from a suspension. Application of the catalyst dramatically improved the oxygen evolution efficiency and corrosion-resistance of stainless steel, nickel and copper anodes in alkaline media. More than 20 g of catalyst was prepared in a single batch, and it was shown to be effective at surface loadings as low as 20 μg/cm 2 . The catalyst was investigated in three different systems: (1) An alkaline electrolyzer with stainless steel electrodes activated with the catalyst supported 120−200% of the current density of an unactivated but otherwise identical electrolyzer, over a range of applied potentials, and maintained this improved efficiency throughout 1495 h of continuous use in 1 M NaOH. (2) Copper anodes were activated and protected from corrosion in dilute sodium hydroxide for 8 h of electrolysis, before a steady decrease in performance over the next 48 h. (3) Activation of nickel anodes with the catalyst reduced the required overpotential by 90−130 mV at current densities between 7.5 and 15 mA/cm 2 , thereby increasing the cell efficiency of water splitting as well as zinc deposition from alkaline zincate electrolytes. The cell efficiency for zinc deposition at a current density of 12.5 mA/cm 2 was improved from 68.0% with a nickel anode to 72.0% with 50 μg/cm 2 catalyst on the nickel anode.

Single-walled carbon nanotubes (SWCNTs) were covalently linked to epidermal growth factor (EGF) proteins through an esterification process that was found to be responsible for the docking of SWCNTs on the human pancreatic cancer cells (PANC-1) surface, thus providing a mechanism for the enhanced delivery and internalization of the nanotubes. Micro Raman spectroscopy and enzyme-linked immunosorbent assay were used to evaluate the delivery process and kinetics of the SWCNTs. In vitro studies indicated that the delivery kinetics of SWCNT-EGF conjugates, at a concentration of 85 µg ml(-1), to the PANC-1 cell surfaces was significant in the first 30 min of incubation, but reached a plateau with time in accordance with the establishment of equilibrium between the association and the dissociation of EGF with the cell receptors. SWCNT-EGF conjugates could act as strong thermal ablation agents and could induce higher percentages of cellular death compared with the nontargeted SWCNTs alone.

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