Abdominal surgical site infections: a prospective study of determinant factors in Harare, Zimbabwe.

Surgical site infections (SSIs) are reported in lower frequencies in the developed countries than in the developing world. A prospective evaluation of risk factors in 285 patients undergoing abdominal surgery procedures in Zimbabwe was therefore undertaken. Overall infection rate was 26%. The age group 30-39 years had the highest number of dirty wounds and the highest rate of human immunodeficiency virus (HIV) infection. Multivariate regression analysis showed a correlation between wound class and SSI (P < 0·05). This was also noted for American Society of Anesthesiologists (ASA) score (P < 0·05). HIV-infected patients had 52% SSIs and non-infected patients had 26% (P < 0·05). Patients receiving blood transfusion had 51% SSIs and those not transfused had 17% (P < 0·01). Patients receiving pre- and intra-operative prophylactic antibiotics had 18% SSIs and those receiving postoperative administration had 37% (P < 0·01). Treatment ranged from dressings only in 11% to surgical intervention in 30% resulting in prolongation of median hospital stay from 8 to 18 days (P < 0·001). Mortality was 7%. High wound class, high ASA score, blood transfusion, HIV infection and delayed use of prophylactic antibiotics were risk factors for SSIs, resulting in surgical interventions, prolonged hospital stay and mortality.

Mechanism-based inactivators as probes of cytochrome P450 structure and function.

The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1.

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.

Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the mutant P450 2E1 T303A.

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P450 2E1 as efficiently with a K(I) of 2.7 microM. The inactivation was entirely dependent on NADPH and followed pseudo-first-order kinetics. Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K(I) of 13 microM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K(i) of 3 microM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of BITC produced characteristic Type I spectral changes in the wild-type and mutant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2E1 T303A when both enzymes were incubated with [(14)C]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consistent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety and one sulfur molecule. Analysis of the metabolites of BITC indicated that each enzyme produced similar metabolites but that the mutant enzyme generated significantly higher amounts of benzaldehyde and benzoic acid when compared to the wild-type enzyme.

Inactivation of cytochrome P450 2B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables.

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time- and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K(I)) was 5.8 microM, and the maximal rate constant for inactivation was 0.66 min(-)(1) at 23 degrees C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 +/- 0. 16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by 2-phenyl-2-(1-piperidinyl)propane.

2-Phenyl-2-(1-piperidinyl)propane (PPP), an analog of phencyclidine, was tested for its ability to inactivate cytochrome P450s (P450s) 2B1 and 2B6. PPP inactivated the 7-(benzyloxy)resorufin O-dealkylation activity of liver microsomes obtained from phenobarbital-induced rats with a K(I) of 11 microM. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified rat liver P450 2B1 and expressed human P450 2B6 was inactivated by PPP in a reconstituted system containing NADPH-cytochrome P450 reductase and lipid. In the presence of NADPH, the loss of activity was time- and concentration-dependent, and followed pseudo first order kinetics. The rate of inactivation for P450 2B1 was 0.3 min(-1), and the concentration of PPP required to achieve half-maximal inactivation was 12 microM. The time for 50% of the P450 2B1 to become inactivated at saturating concentrations of PPP was 2.5 min. P450 2B6 was inactivated with a k(inact) of 0.07 min(-1), a K(I) of 1.2 microM, and a t(1/2) of 9.5 min. The inactivated P450s 2B1 and 2B6 lost about 25 and 15%, respectively, of their ability to form a CO-reduced complex, suggesting that the loss of activity was caused by a PPP modification of the apoprotein rather than the heme. The estimated partition ratio for P450s 2B1 and 2B6 with PPP was 31 and 15, respectively. The inactivation was not reversible and reductase activity was not affected. Coincubation of P450 2B1 and 2B6 with PPP and NADPH in the presence of an alternate substrate protected both enzymes from inactivation. The exogenous nucleophile GSH did not affect the rate of inactivation. PPP-inactivated P450s 2B1 and 2B6 were recognized on Western blots by an antibody generated to phencyclidine that had been conjugated to BSA. Stoichiometries of 1.4:1 and 0.7:1 were determined for the binding of a [3H]PPP metabolite to P450 2B1 and 2B6, respectively.

Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates.

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy-ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.

Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass spectrometry.

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) O-deethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min(-)(1) at 30 degrees C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t(1/2)) was 1.8 min. The estimated concentration which led to half-maximal inactivation (K(I)) was 25 microM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentration-dependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.

Inactivation of cytochrome P450 2E1 by benzyl isothiocyanate.

The cytochrome P450 enzymes constitute a family of phase I enzymes that play a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds. In this study, the kinetics for the inactivation of cytochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broccoli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified and reconstituted P450 2E1 in a time- and concentration-dependent manner. The concentration of inactivator needed for half-maximal inactivation (K(I)) was 13 microM, and the maximum rate of inactivation at saturation (k(inact)) was 0.09 min-1. The partition ratio for the inactivation of P450 2E1 by BITC was found to have an approximate value of 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activity was observed, while approximately 74% of the spectrally detectable enzyme remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P450 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation activity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inactivation of P450 2E1 by BITC was irreversible, and no activity was regained after extensive washes to remove BITC. Addition of cytochrome b(5) to the reconstituted system did not affect the rate of inactivation. Reductase activity was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivation was primarily due to a modification of the apoprotein by BITC.

Selective mechanism-based inactivation of cytochromes P-450 2B1 and P-450 2B6 by a series of xanthates.

Fifteen xanthates with carbon chains of different lengths or substitutions, including the antiviral compound D609 (O-tricyclo[5.2. 1.0(2,6)]dec-9-yl-dithiocarbonate), were tested for their ability to inactivate cytochromes P-450 (P-450s) 2B1 and 2B6. All of the xanthates tested were found to inactivate P-450 2B1 in a time- and concentration-dependent manner. The rates of inactivation at 30 degrees C ranged from 0.22 min-1 to 0.02 min-1. The concentrations required for half-maximal inactivation were between 2.4 and 69 microM. A general trend in the inactivation kinetics could be observed with an increasing chain length of the xanthates. Longer carbon chains resulted in slower rates of inactivation with longer half-times of inactivation and higher partition ratios. For P-450 2B1, the most effective inactivators were xanthates with substitutions of intermediate length. The best inactivator for P-450 2B1 was the C8 xanthate, with an inactivation potency (KI) of 2.4 microM, a rate of inactivation of 0.07 min-1, and a partition ratio of 4. Four xanthates were further examined for their effect on the 7-ethoxy-4-(trifluoromethyl)coumarin activity of P-450 2B6. The C8 xanthate was again the most effective inactivator, with a KI of 1 microM. Although the KI values were generally lower than those found with P-450 2B1, the rates of inactivation for P-450 2B6 with the various xanthates were 3- to 5-fold slower. In addition, the isozyme selectivity of xanthates was tested with P-450s 2E1, 1A1, 3A2, 3A4, 2C9, and 2D6. P-450 2E1 was inactivated by xanthates at concentrations 15- to 100-fold higher than those required to inactivate either P-450 2B1 or 2B6. P-450 1A1 was not inactivated by xanthates. However, all of the xanthates tested were able to inhibit the enzymatic activity of P-450 1A1 to a different extent, depending on the length of the xanthate carbon chain. Virtually no inactivation of P-450s 2D6 or 2C9 was seen, except that C8 and D609 were inhibitory at high concentrations (0.2-0.6 mM). None of the xanthates studied had any effect on the activities of P-450s 3A2 or 3A4.

Microsomal cytochromes P450 catalyze the oxidation of low density lipoprotein.

Low density lipoprotein (LDL) oxidation is a major contributor to foam cell formation during early atherogenesis. Several oxygenases have been implicated in the process of LDL oxidation in the arterial wall, where the environment is relatively low in antioxidants, but the exact mechanism for LDL oxidation in vivo is not known. In the present study we sought to determine the ability of cytochrome P450 2E1 (P450 2E1) and other P450s, located in the liver and in other tissues, to oxidize LDL. Upon incubation of LDL (0.1 mg of protein/ml) with purified, reconstituted rabbit P450 2E1 in the presence of NADPH and the NADPH-cytochrome P450 reductase, time- and P450 2E1 concentration-dependent LDL oxidation was observed, as analyzed by determining the formation of peroxides, thiobarbituric acid reactive substances (TBARS), and conjugated dienes. Within 1 h of initiating the reaction, almost maximal oxidation was observed. NADPH, and active P450 2E1 enzyme were required for LDL oxidation to occur. The rate of P450 2E1-induced LDL oxidation was also dependent on the lipoprotein concentration. P450 2E1 could also oxidize pure phospholipids and cholesteryl ester, the major lipids in LDL. In the presence of catalase or superoxide dismutase (SOD), LDL oxidation was completely blocked, suggesting that hydrogen peroxide and superoxide are involved in P450 2E1-induced LDL oxidation. The ability of P450 2E1 to oxidize LDL was not unique to this enzyme, and could be observed with some other purified, cytochromes P450 in the reconstituted system such as rat P450 2B1 and human P450 3A4. Finally, microsomal membranes obtained from rats that were induced to express high levels of P450s 2B1, 2E1, and 1A1/2 were able to oxidize LDL, whereas little oxidation was seen with microsomes that were induced to express 3A2. We thus conclude that LDL can be oxidized by some cytochrome P450s and, as some of these enzymes are present in liver and in arterial wall, they may have a physio/pathological relevance to LDL oxidation and atherogenesis.

Mechanism-based inactivation of cytochromes P450 2B1 and P450 2B6 by n-propylxanthate.

n-Propylxanthate (nPX) inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified, reconstituted rat hepatic P450 2B1 or human P450 2B6 in a mechanism-based manner. The inactivation followed pseudo-first-order kinetics and was entirely dependent on both NADPH and nPX. The maximal rate constant for inactivation of P450 2B1 at 30 degrees C was 0.2 min-1. The apparent KI was 44 microM, and the half-time for inactivation was 4.1 min. Purified, reconstituted human P450 2B6 was also inactivated by nPX with a KI of 12 microM. The kinactivation for P450 2B6 was 0.06 min-1, and the t1/2 was 11 min. Incubations of P450 2B1 with nPX and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 25% loss of the enzyme's ability to form a reduced CO complex. Little loss in the absolute spectrum of nPX-inactivated P450 2B1 was observed. With P450 2B6, an 83% loss in enzymatic activity and a 12% loss in the CO-reduced spectra were observed. The extrapolated partition ratio for nPX with P450 2B1 was 32. P450 2B1 could be protected from inactivation by nPX by adding an alternate substrate to the reaction mixture. Removal of unbound nPX by dialysis did not reverse the inactivation. The alternate oxidant iodosobenzene was able to partially restore enzymatic activity to nPX-inactivated P450 2B1 samples. A stoichiometry for labeling of 1.2:1 for binding of radiolabeled nPX metabolite to P450 2B1 was seen. These results indicated that nPX inactivated P450 2B1 and P450 2B6 in a mechanism-based manner. P450 2B1 was inactivated primarily by a nPX reactive intermediate that bound to the apoprotein.

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate.

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.

Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1.

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.

Mechanism-based inactivation of cytochrome P450 2B1 by N-benzyl-1-aminobenzotriazole.

The kinetics of inactivation of cytochrome P450 2B1, the major phenobarbital inducible rat hepatic P450, by N-benzyl-1-aminobenzotriazole (BBT) were characterized. Purified, reconstituted P450 2B1 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylase activity was inhibited by BBT in a mechanism-based manner. The loss of O-deethylase activity followed pseudo-first-order kinetics and was NADPH and BBT dependent. After a 5 min incubation, greater than 90% of the 2B1 activity was lost, whereas more than 70% of the ability of the reduced enzyme to bind CO was maintained. Inclusion of 10 mM glutathione in the inactivation reaction lowered the rate of inactivation (k(inactivation)) and increased the partition ratio without significantly affecting the inactivator concentration required for half-maximal inactivation (K(I)). The maximal rate constant for inactivation at 23 degrees C was 0.24 min(-1) without and 0.15 min(-1) with glutathione. The apparent K(I) was 2 microM in both cases. The extrapolated partition ratios were 4 and 9 without and with 10 mM glutathione, respectively. Consistent with mechanism-based inactivation, the loss of 7-EFC O-deethylase activity was irreversible, was not due to product inhibition, was saturable, and could be slowed by including increasing concentrations of competing substrate. However, the inactivated P450 2B1 was still able to metabolize substrate if iodosobenzene was used as an alternate oxidant. Inactivation of 2B1 with either N-[14C]-7-benzyl-1-aminobenzotriazole (BBT) or N-benzyl-1-amino-[14C]-2,3-benzotriazole resulted in the incorporation of covalent radiolabel into the apoprotein. The stoichiometry of labeled metabolite adduct to protein was approximately 0.4:1 in both cases. Identification of metabolites revealed the formation of 1-aminobenzotriazole, benzotriazole, benzaldehyde, and a new metabolite (27) during catalysis of BBT by P450 2B1. Together, these data suggest that P450 2B1 could be inactivated and labeled by more than one metabolite.