The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC 50 s) of ϳ2 and ϳ6 M, respectively. Cyclosporin A was inhibitory with an IC 50 of 1.4 M in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the K m values were congruent with the IC 50 s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport.

Many catalytic activities of cytochrome P450 (P450)3A4 More than 40 P4501 enzymes are found in a single mammalian species (2). The proteins constitute a superfamily and collectively contribute extensively to the oxidation of xenobiotic chemicals (e.g. drugs, carcinogens, pesticides, alkaloids, and other natural products) and also endobiotics (e.g. steroids, eicosanoids, fat-soluble vitamins, fatty acids) (3-6). The contributions of these P450 enzymes to metabolism in humans are well recognized, particularly regarding issues of drug clearance (7-9). There is general agreement that, in most humans, P450 3A4 is the most abundant of the P450s in both liver and small intestine (8, 9); it can constitute up to 60% of the total P450 in the liver (10). The intestinal enzyme has been implicated in variation in the bioavailability of many orally administered drugs (11). P450 3A4 has a very broad range of substrates, with more than 60 drugs having been already identified (9). These vary widely in structure, and one of the questions about this enzyme has been the molecular basis of its broad catalytic specificity (12, 13). Other mechanistic questions involve the basis of the sigmoidal plots of enzyme velocity versus substrate seen with some compounds (14 -16) and the stimulation of activity by chemicals other than the substrate (14,17,18). The purified enzyme, along with other P450 3A subfamily enzymes, is much more sensitive to its reconstitution environment than are most other P450s (19, 20). A variety of components have been reported to stimulate catalytic activity, including long chain unsaturated phosphatidylcholines (21), phosphatidylserine (20,22), ionic detergents (21, 22), GSH (23), divalent cations (24, 25), and b 5 (19,21). Not all of these components are directly relevant to the membrane-bound enzyme, but Mg 2ϩ has been shown to stimulate activity of the enzyme in microsomes (24, 25) and antibodies raised against b 5 can inhibit some catalytic activities of P450 3A4 in microsomes (19,25). Somewhat surprisingly, certain catalytic activities of P450 3A4 are quite refractory to alterations in lipids and b 5 (24, 26).In order to better understand this complex but important system, we initiated a systematic investigation of some of the system components on individual steps in the catalytic cycle of purified recombinant P450 3A4 (14,19,20,(23)(24)(25). A general conclusion about the role of b 5 in modulating P450 reactions has been that electron transfer from b 5 to P450 occurs in step 4 of Scheme 1 (27). Recently we found qualitative evidence that b 5 could also stimulate the reduction of P450 3A4 by the flavoprotein NADPH-P450 reductase (24). We now report that apo-b 5 (devoid of heme) can replace b 5 in the efficient oxidation of the prototypic P450 substrates testosterone and nifedipine and that apo-b 5 can also replace b 5 in the facilitating electron flow from NADPH-P450 reductase to P450 3A4, in the absence of electron transfer from b 5 or modulation of the E m,7 of P450 3A4.

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Aflatoxin B1 (AFB1) is thought to play a large role in human liver cancer in some parts of the world, and the mechanism of genotoxicity is generally considered to involve the DNA adduct formed at the guanyl N7 atom. The exo epoxide, the genotoxic isomer formed by human cytochrome P450 3A4, has been known to be very unstable in H2O (t 1/2 < 10 s). The rates of hydrolysis of AFB1 exo epoxide have been determined as a function of pH using stopped flow kinetics. The spontaneous reaction with solvent is faster than previously suspected, with a t 1/2 ∼1 s when either absorbance or fluorescence kinetic traces are measured at ambient temperature. An acid-catalyzed reaction with a bimolecular rate constant of 2 × 103 M-1 s-1 is operative below about pH 5 and elevates the rate. The hydrolysis product, AFB diol, reversibly converts to a furofuran-ring-opened oxyanionic AFB1 α-hydroxydialdehyde (AFB dialdehyde) under slightly basic conditions. When AFB diol was treated with base in DMSO, conversion to AFB dialdehyde was accompanied by dehydration of the remaining alcohol group. In aqueous solution, the AFB diol:AFB dialdehyde equilbrium has been characterized and the pK a (8.2) is much higher than previously suggested; the base-catalyzed bimolecular rate of 2.3 × 103 M-1 s-1 results in a very slow rate of conversion at physiological pH. Multiphasic conversion of AFB dialdehyde back to AFB diol has a slow rate-limiting step at 0.01 s-1 and is nearly quantitative. The dehydrated AFB dialdehyde formed in DMSO did not form a detectable ring closed derivative.