Chem. Res. Toxicol. 1991,4, 408-413
408
Articles Deuterium Isotope Effect in the Interaction of N-Nitrosodimethylamine, Ethanol, and Related Compounds with Cytochrome P-450 I IE l Chung S. Yang,* Hiroyuki Ishizaki, Maojung Lee, David Wade,+and Addi Fade1 Laboratory for Cancer Research, Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855-0789 Received November 15, 1990 Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity and produces an isotope effect on its metabolism. Our previous results showed that deuteration causes a 5-fold increase in the apparent K,, but not the V-, for the demethylation and denitrosation of NDMA in microsomes. In the present work, we studied the nature of this deuterium isotope effect with several compounds using acetone-induced microsomes as a source of cytochrome P-450IIE1. In the microsomal N-nitrosodiethylamine deethylase reaction, NDMA and [2H6]NDMA were competitive inhibitors and displayed apparent Ki values of 59 and 441 mM, respectively, showing an isotope effect of 0.13. Similarly, in the p-nitrophenol hydroxylase reaction, a deuterium isotope effect of 0.21 on the Ki was observed. With acetone as an inhibitor for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. When the oxidation of ethanol, [ 1,1-2H2]ethanol,[2,2,2-2H3]ethanol,and [2H6]ethanolwas compared, an isotope effect of about 5 was found in the V - / K , due to the deuteration of the methylene group (carbon 1)but not due to the methyl group. However, the V,, was not affected. A corresponding deuterium isotope effect was observed in the Ki when these compounds were used as competitive inhibitors for the NDMA demethylase reaction. The results demonstrate that deuteration of NDMA, ethanol, and related compounds results in an increase in the K, or Ki with little change in the V,, of P-450IIEl-catalyzed reactions. The molecular basis of this isotope effect is discussed.
Introduction N-Nitrodimethylamine (NDMA),' a potent and widely occurring carcinogen, requires a cytochrome P-450(P-450) dependent monooxygenase for ita activation (1-5). Keefer et al. (6) found that the potency of the fully deuterated analogue, [2H6]NDMA,was much lower than that of undeuterated NDMA and attributed the result to a difference in the metabolism of these two compounds. Concerning the nature of the isotope effect, Dagani and Archer (7) found that deuteration caused a reduction in the V,, and K, for the demethylation of high concentrations of NDMA by liver microsomes from phenobarbital-treated rats. Using low substrate concentrations and rat liver S-9 fraction, Kroeger-Koepke and Michejda (8) also observed deuterium isotope effects on both the V,, and K, for the demethylation of NDMA. Swann et al. (9) found a small isotope effect on the in vivo conversion of NDMA to COz when NDMA and [2Hs]NDMAwere administered separately, but a larger effect when they were administered simultaneously. From this result, they concluded that carbon-hydrogen bond cleavage was not a rate-limiting step in the in vivo metabolism of NDMA but rather that deuterium substitution affected the equilibrium binding of NDMA to the enzyme *Towhom correspondence should be addressed. 'Present address: Rockefeller University, New York, 10021.
active site. When low doses of NDMA were given to rats, Mico et al. (10) found an isotope effect on the rate of NDMA elimination from the blood, and the effect was more pronounced when the two isotopic analogues were administered simultaneously. These observations are important in understanding the mechanism of activation of this carcinogen in vivo. The nature of the kinetic isotope effect reported for studies in vitro (7, 81, however, is puzzling and inconsistent with the results from studies in vivo. Previous work from this laboratory and others has demonstrated that the low K , form of NDMA demethylase is manifested by P-450IIE1 (also known as P-450ac, P-450j, or P-450LMa) and that this enzyme is responsible for the metabolism and activation of low concentrations of NDMA (4,5,11-14). With acetone-induced microsomes, we found that deuteration of NDMA increased the apparent K , of the demethylation and denitrosation by 5-fold but did not change the V,, of the reaction (15). This observation is consistent with the results obtained in studies in vivo (9, 10) but is also unexpected. In most known examples, deuterium substitution Abbreviations: NDMA, N-nitrosodimethyGine; P-460,cytochrome P-450;[*Ha]NDMA,N-nitrosodi([lfI&uethyl)amine (other deuterated compounds are abbreviated similarly);NDEA, N-nitrosodiethylamine; DMF, dimethylformamide;HPLC,high-performanceliquid chromatography.
0893-228~/91/2704-0408$02.50/0 0 1991 American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. 4, 1991 409
Isotope Effect on K,,, and Ki
Table I. Deuterium Isotope Effect on Kinetic Parameters of P-450IIEl-Catalyzed Reactionsa Vmam
expt 1
2
3 4
5
inhibitor (pM) none NDMA (100) [2H6]NDMA(525) none NDMA (50) [2H6]NDMA(107) none acetone (2000) [2H6]acetone(2000) none acetone (1000) [2H6]acetone(5000) none DMF (500) [2H,]DMF (1000)
substrate NDEA NDEA NDEA p-nitrophenol p-nitrophenol p-nitrophenol p-nitrophenol p-nitrophenol p-nitrophenol NDMA NDMA NDMA NDMA NDMA NDMA
n 5 5 5 8 5 6 5 4 4 4 4 4
3 3 3
nmobmin-'. mg-' 4.77 f 0.88 4.24 f 0.60 4.67 f 0.83 5.96 f 0.43 5.53 f 0.59 5.73 f 0.58 6.02 f 0.10 5.39 f 0.28 5.54 f 0.54 7.25 f 0.17' 6.63 f 0.25 6.40 f 0.27 7.30 f 0.91 6.95 f 1.22 5.97 f 0.29
K',, rM 42 f 8 117 f 23 100 f 24 14 f 3 33 f 9 23 f 4 12 f 2 56 f 4 18 f 4 23 f 3 78 f 9 45 f 5 23 f 3 205 f 28 120 i 13
Ki, rM 59 f 12 441 f 180b 46 f 20
isotope effect on Ki 0.13 0.21
214 f 152b
484 f 78 4322 f 684*
0.11
456 f 149 4395 f 260b
0.10
66 f 16 245 f 51b
0.27
OThe results are expressed as mean f SD, and n equals the number of experiments. The concentrations of the inhibitors are shown in parentheses. The apparent V,, and K, values were obtained by nonlinear regression. The differences in the means were analyzed by the Student-Newman-Kuels procedure. The Ki was calculated from each set of experiments. Significantly different 0, < 0.05) from the result obtained with the nondeuterated analogue by the Student t test. 'Significantly different 0, < 0.05) from the results obtained in the presence of inhibitors.
has been shown to reduce the rate of the carbon-hydrogen bond cleavage and to result in a decrease in the V,, value (16, 17).
In order to study the nature of the deuterium isotope effect in the interaction between P-450IIE1 and its substrates, NDMA, acetone, dimethylformamide (DMF), and ethanol and their deuterated analogues were used as competitive inhibitors for the metabolism of other P-450IIE1 substrates. Isotopomers of ethanol deuterated at the methyl and methylene groups were used to differentiate the origin of the deuterium isotope effect.
Experimental SectIon Chemicals. NDMA, N-nitrosodiethylamine (NDEA), p nitrophenol, 4-nitrocatechol, DMF, and deuterated ethanol analogues were obtained from Aldrich Chemical Co. (Milwaukee, WI). NADP, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, [2Hs]acetone, and [2H7]DMFwere from Sigma Chemical Co. (St. Louis, MO). Ethanol was obtained from Pharmco Products, Inc. (Bayonne, NJ). [2H6]NDMAwas synthesized (6)and kindly provided by Dr.Larry K. Keefer (National Cancer Institute, Frederick, MD). The minimum isotopic purity of the deuterated compounds was >99.5%. The chemical purities of the deuterated compounds and their nondeuterated analogues were >99%. Microsomes. Male Sprague-Dawley rats, from Taconic Farms (Germantown, NY), with body weights of approximately 100 g were used. For the induction of P-450IIE1, the rats were treated with one dose (intragastrically, 5 mL/kg body weight) of acetone 22 h before sacrifice. Liver microsomes were prepared as described previously (18). The acetone-induced microsomes were found to be a suitable system for studying the catalytic activities of P450IIE1. These microsomes display much lower K , values for NDMA demethylase (19, 20) than purified P-450IIE1 in a reconstituted system (12) because of the presence of glycerol and probably other competitive inhibitors in the latter system. In experiments using low substrate concentrations,antibodies against P-450IIE1 almost completely inhibit the NDMA demethylase activity, suggesting negligible contributions from other P-450 forms (20, 21 ). NDMA Demethylase Assay. All reactions were run in duplicate in borosilicate glass test tubes. The reaction mixture contained 50 mM Tris-HC1 (pH 7.4), 10 mM MgCIP,150 mM KCl, and an NADPH-generating system (0.4 mM NADP, 10 mM glucose 6-phosphate, and 0.2 unit of glucose-6-phosphate dehydrogenase), microsomes corresponding to 0.06 mg of protein, and NDMA ranging from 40 to 600 pM in a total volume of 0.25 mL. The reaction was initiated by the addition of NDMA after a preincubation at 37 OC for 1-2 min. After an incubation period
of 10 min at 37 OC, the reaction was terminated by the addition of ZnSO, and Ba(OH)2solutions, and the formaldehyde produced was determined by the Nash reaction as described previously (19, 22). Blanks and standards were run simultaneously with the omission of the substrate. Substrates and inhibitors, a t the concentrationsused, had no effect on the absorbance. The present assay conditions differed slightly from those used previously (15), and lower K , values were observed. NDEA Deethylase Assay. The incubation conditions were similar to the NDMA demethylase assay, containing 0.075 mg of microsomal protein and NDEA ranging from 20 to 320 pM in a total volume of 0.5 mL. The test tubes were capped with rubber stoppen to minimize the loss of acetaldehyde through evaporation. After an incubation for 15 min, the reaction was terminated by the injection of 0.1 mL of a mixture containing 17% ZnSO, and 0.55 mM semicarbazide which serves to trap acetaldehyde. The mixture was deproteinized, and the acetaldehyde was analyzed as a (2,4-dinitrophenyl)hydrazone derivative by the HPLC as described previously (23, 24). Ethanol Oxidase Assay. The conditions were similar to the NDEA deethylase assay by measuring the acetaldehyde formed except 0.12 mg of microsomal protein, 0.1-8.0 mM of ethanol, and a 10-min incubation time were used. p-Nitrophenol Hydroxylase Assay. The hydroxylation of p-nitrophenol was determined according to the method of Reinke and Moyer (25). The incubation conditions were similar to those of other assays. The reaction mixture (0.5 mL) contained 0.12 mg of microsomal protein and l e 5 0 mM p-nitrophenol, and the reaction was initiated by the addition of the NADPH-generating system. After an incubation at 37 OC for 6 min, the reaction was terminated by the addition of 0.25 mL of 0.6 N HClO,. After centrifugation, 0.5 mL of the supernatant was mixed with 0.05 mL of 10 N NaOH. AWm was measured for the determination of 4-nitrocatechol. Analysis of the Results. All the data points in each experiment were the average of two duplicated determinations. Mean substrate concentrations (26) were used, and the data were analyzed by the Lineweaver-Burk (double-reciprocal) plot and a nonlinear regression method (Enzfitter, Elsevier: B I ~ F T ) .T h e results from the second method of analysis are presented in Table I. Apparent Ki values were calculated for each set of experiments by using the equation Ki = K,[I]/(K', - Km), where K, and K6, were obtained in the absence and the presence of inhibitor, respectively, and [I] is the concentration of the inhibitor.
Results Inhibitory Action of NDMA and [*H,]NDMA on the Metabolism of NDEA and p-Nitrophenol. Because of the structural similarity, NDMA was expected to be a competitive inhibitor of the metabolism of NDEA. The
Yang et al.
410 Chem. Res. Toxicol., Vol. 4, No. 4, 1991 0.8
2.5 A
w
E .
2.0
0.6
-
E
3 'E. 1.5
0
E
.
1.o
C
I
Y
l-
0.5
0.0'
0
'
1 0
.
20
3 0
40
50
1/[NDEA,mM]
'
60
0.0
0
I
1
1 0
20
30
l/[NDMA,mM]
Fuure 1. LineweaverBurk plots showing competitive inhibition. Panel A Inhibition of NDEA deethylase by NDMA and [NINDMA. symbols: ( 0 )no inhibitor; (4 [WINDMA(525 pM);and (D) NDMA (100 pM).Panel B Inhibition of NDMA demethylase by acetone and [wacetone. Symbok (0)no inhibitor; (A)[%]acetone (4mM); and (D) acetone (2 mM). The data are from one set of experiments with duplicate assays.
deuterium isotope effect on the inhibitory action is presented in double-reciprocal plots (Figure lA), and the results of 5 sets of experiments are summarized in Table I. Both NDMA and [2H6]NDMAshowed characteristics of competitive inhibitors in the NDEA deethylase assay. Because [2H6]NDMAwas a much weaker inhibitor than NDMA, a higher concentration was used in order to obtain accurate data. The apparent V,, values of the three reactions were not statistically different. On the basis of the competitive inhibition model, the calculated apparent K i values, 59 and 441 pM for NDMA and [2H6]NDMA, respectively, represent an isotope effect of 0.13 (Table I). Similar results were observed when another substrate of P-450IIE1, p-nitrophenol (27),was used as a substrate (Table I). The presence of NDMA and [2H6]NDMAdid not affect the V,, of the p-nitrophenol hydroxylase reaction, but an isotope effect of 0.21 was observed in the Ki values of these two competitive inhibitors. The result suggested that replacing the 6 protium atoms of NDMA with deuterium atoms decreases its effectiveness in serving as a competitive inhibitor in P-45OIIEl-catalyzed reactions. Effects of Deuterium Substitution on the Inhibitory Actions of Acetone and Dimethylformamide. Acetone and DMF were found to be competitive inhibitors of the NDMA demethylase reaction in previous studies (19). In the p-nitrophenol hydroxylase assay, both acetone and [%]acetone were competitive inhibitors and displayed K i values of 484 and 4322 pM, respectively, showing a kinetic isotope effect of 0.11 (Table I). Both acetone and [2H6]acetonewere competitive inhibitors of NDMA demethylase, and the former was more potent (Figure 1B). In 4 sets of experiments (Table I) acetone and [2H6]acetone displayed apparent Ki values of 456 and 4395 pM, respectively, reflecting a deuterium isotope effect of 0.10. In these experiments, the apparent V,, values in the presence of inhibitors were slightly lower than those in the absence of the inhibitor, possibly due to the denaturation of enzyme in the presence of a rather high concentration of acetone. DMF and [2H,JDMF were also competitive inhibitors of NDMA demethylase with apparent Ki values of 66 and 224 pM, respectively (Table I), demonstrating an isotope effect of 0.27. In these studies, deuterated acetone and DMF were used in higher concentrations than their protium isomers in order to obtain data that could be accurately analyzed. Similar kinetic parameters were also obtained when 2 mM acetone and [2H6]acetonewere used as well as when 1mM of DMF and [2H,]DMF were used. In another experiment, DMF was incubated with
Table 11. Separation of ['H'INDMA from NDMA by High-Performance Liquid Chromatographp retention time, min system elution solvent [2He]NDMA NDMA 1 0.1% methanol 25.1 26.0 2 0.1% methanol, pH 3.6 48.2 50.0 3 water 57.5 60.0 O A Waters pBondapak CI8 (lO-pm), 30 cm X 3.9 mm column was used in systems 1 and 3; two columns were used in series in system 2. The flow rate was 0.5 mlsmin-' except for system 3, at 0.2 ml-min-'. The amounts of NDMA and [2H,]NDMA used in each experiment were from 200 to 300 nmol, and they were monitored at 254 nm. A Waters Model 510 pump and Model 490E detector were used for the experiment.
microsomes in the presence of an NADPH-generating system for 20 min to determine whether DMF could serve as a substrate for P-45OIIE1. Disappearance of substrate or formation of a possible product, HCHO, was not observed (data not shown). Difference in Lipophilicity between NDMA and [2H6]NDMA. In order to determine the effect of deuterium substitution on the lipophilicity of NDMA, NDMA and [NlNDMA were subjected to reversed-phase HPLC. In three systems of elution solvent, [%]NDMA was found consistently to be eluted ahead of NDMA (Table 11), suggesting that [2H6]NDMAis less lipophilic or hydrophobic than its undeuterated analogue. Metabolism and Inhibitory Action of Ethanol Isotopomers Deuterated at Different Positions. In order to further characterize the nature of the presently observed isotope effect, ethanol isotopomers deuterated at the methylene and methyl groups were analyzed. If the isotope effect is due to a decreased affinity of the deuterated compounds in the reversible binding to the active site of the enzyme, then the magnitude of the effect due to the extent of substitution should be [2H6]ethanol > [2,2,2%]ethanol 1 [1,1-2H2]ethanol. If the breaking of the C-H bond is the major factor for the isotope effect, then the effect should be observed only when the methylene group is deuterated. The results in Figure 2 and Table I11 favor the second possibility. When the methylene group was deuterated, an isotope effect was observed in the K , but not in the V,, (Figure 2). The results in Table I11 demonstrated that an isotope effect of 0.14-0.20 on the K , (or about 5 for V-lK,) was produced by the deuteration at the methylene group, but not at the methyl group. Deuteration at any position did not affect the V - . When
Chem. Res. Toxicol., Vol. 4,No.4,1991 411
Isotope Effect on K,,, and Ki Table 111. Deuterium Isotope Effect on the Oxidation of Ethanol'
substrate ethanol [l,l-zH2]ethanol [ 2,2,2JH9]ethanol [zHB]ethanol
n 3 3 3
1.6
isotope v,, effect nmol. min-lemg-' K,, pM VIK K, VIK 5.32 f 0.96 204 f 70 26.1 6.77 f 0.96 1409 f 100b 4.8 0.14 5.4 5.55 f 0.81 222 f 91 25.0 0.92 1.1
-
. .0)
1.2
E E
E -. 0.8 0
3 5.38 f 1.58 1032 f 40b
5.2 0.20
E
5.0
=.
-.
'The results are expressed as mean f SD, and n equals the number of experiments. The apparent K, and V,, values were obtained by nonlinear regression. The K, and V,, values for each group of three sets of experiments were analyzed by the Student-Newman-Kuels procedure. bSignificantly different from that with nondeuterated ethanol and from each other (p < 0.05).
L
"." 0
ethanol and deuterated analogues were used as inhibitors in the NDMA demethylase assay, again, an isotope effect of 0.15-0.17 on the Ki was observed with the methylenedeuterated compounds (Table IV). When the methyl group is deuterated, an isotope effect of 0.54 on the Ki was observed, but the statistical significance and the nature of this effect are not known. Secondary isotope effects may be a factor in this situation.
2
4
6
8
14
(A) [l,l-2H2]ethanol.
zyme-substrate complex. The results in Table I1 showing a decreased hydrophobicity in the deuterated analogue appear to support the second possibility. However, it is questionable whether such a small change in hydrophobicity could result in severalfold changes in the Ki. The deuterium isotope effects on the oxidation of ethanol were studied previously by Ekstrom et al. (29),and an isotope effect of 4 in the V,,/K, was observed when the methylene group was deuterated. However, from these data, it is not possible to determine whether there was an isotope effect on the V-. The results in Table I11 clearly demonstrate that the kinetic isotope effect was on the K, but not on the Vm. Furthermore, the effect was observed when the methylene group, but not the methyl group, was deuterated. The results can also have two interpretations: (a) The observed deuterium effect is due to the rate difference in the breaking of the C-H bond and its relationship with other rate constants. (b) It is due to a change in the affinity of the reversible binding after deuterium substitution. This binding effect may be manifested if there is hydrogen bonding between the OH group of ethanol and an amino acid residue in the active site of P450IIE1. The hydrogen bonding may be weakened by the deuteration of the adjacent carbon 1but not significantly by the deuteration of carbon 2. However, it is questionable whether such a secondary isotope effect can be responsible for the rather large effect observed herein. Evidence for hydrogen bonding between P-450IIE1 and substrate is lacking. The results that (2)and (E)-[2H3]NDMAdisplayed the same isotope effect on the K , in the demethylation reaction and that almost all the formaldehyde formed was derived from the nondeuterated methyl group (28) agree with the concept that the rate of the carbon-
Table IV. Deuterium Isotow Effect on Inhibitory Action of Ethanol' substrate n V-, nmol.min-'.mg-' K',, pM K,,pM 3 3 3 3 3
12
1 I [S,mM]
Dlscusslon
NDMA NDMA NDMA NDMA NDMA
10
Figure 2. Lineweaver-Burk plots of the rate of acetaldehyde formation from ethanol oxidation. The data are from one set of experiments with duplicate assays. Symbols: ( 0 )ethanol and
Isotopic substitution, which is considered not to alter the electronic potential energy surfaces of molecules, has been used extensively to address questions concerning mechanisms in enzymology. Upon substitution of deuterium for protium, most investigators have observed a substantial rate reduction in reactions that involve a proton-transfer step (16,17).In our previous studies with NDMA, [2H3]NDMA,and [2H,]NDMA, however, an isotope effect was not observed on the V-, but on the K, or V&K, (15,B).In the present work, an isotope effect of 0.10-2.7 was observed in the apparent Ki values when deuterated and nondeuterated NDMA, acetone, ethanol, and DMF were used as competitive inhibitors for different P-450IIEl-catalyzed reactions. Since the Ki value was calculated from each experiment on the basis of the difference between K & and K,, experimental errors could have been amplified as seen in the rather large standard deviations in some experiments. However, the apparent Ki values for all the deuterated compounds were significantly different (at p < 0.05) from those for their nondeuterated analogues. The aforementioned deuterium isotope effect may be due to (a) the difference in the rate of the carbon-hydrogen bond cleavage and its relationship with other rate constants or (b) the difference between NDMA and [2Hs]NDMA in the affinity of binding to the active site of P450IIE1. If the inhibitor is a competitive substrate, then the Ki is equivalent to the K,; otherwise, the Ki may be equivalent to the K,, the dissociation constant of the eninhibitor (mM) none ethanol (0.2) [ l,l-*H,]ethanol (1.0) [2,2,2-2H3]ethanol(0.2) [2He]ethanol(1.0)
0.4
4.52 f 0.5 5.03 f 0.5 3.96 f 0.1 3.45 f 0.8 3.62 f 0.2
14 f 2 104 f 95 44 f 6 47 f 10 40 f 3
77 f 17 451 f 96b 143 f 54 524 f 133b
isotope effect on Ki 0.17 0.54 0.15
'The incubation conditions were similar to those in Table I, except that ['FINDMA (10-100 pM) and acetone-induced microsomes (0.025 mg of protein) were used. After an incubation for 10 min, the reaction was terminated and the H"CH0 formed was determined as a dimedone adduct (29). Because of the lower substrate concentrations and amount of microsomes used, the K, value of NDMA demethylase was lower than those in Table I. The differences in the means of V,, and Ki values were analyzed by the Student-Newman-Kuels procedure. *Significantly different @ < 0.01) from that of the nondeuterated ethanol.
Yang et al.
412 Chem. Res. Toxicol., Vol. 4, No. 4, 1991
hydrogen bond cleavage is the major factor in determining the deuterium isotope effects observed in this work. It is not clear how the rate of carbon-hydrogen bond cleavage affects the K, or VIK but not the V-. It may be interpretable according to a scheme recently proposed by Gillette and Korzekwa (30),as shown: NADP+ t
S t E
kl2 e kz 1
In this scheme, the substrate combines reversibly with enzyme to form an enzyme-substrate complex (ES). The ES complex is then reduced, oxygenated, and further reduced to form a dioxygenated complex (EOa). The EO# complex can be converted to an oxenoid complex (EOS) or decompose to form ES complex and HzOz. In the EOS complex, the substrate is oxidized to form an enzymeproduct complex (EP) which then dissociates to form the product and regenerate free enzyme. The isotopically sensitive step is the breaking of the C-H bond with rate constant k46. According to Northrop (31),when a single product is formed and the isotopically sensitive step is preceded by an irreversible step, the apparent isotope effect on V,,/K, should be 1.0. However, an isotope effect can be observed if the EOS complex can be reduced by NADPH to form ES and HzO (with rate constant k42). In such a case
and D(V) =
k46H/k46D
+
1+R
where
and
R=
IZ46Hfk34
+ k32 + k23 + (k2&34/k61)1
+ k32 + k23)k42 + k23k34 The C and R bear the same meanings of “commitment to catalysis”and “ratio of catalysis”, respectively, as discussed by Northrop (31). In order to have an isotope effect on V,,/K, but not on V,, C has to be very small and R has to be very large. This condition can be satisfied if k 4 2 is much larger than k46H, and is much smaller than kaka. The formation of water at the expense of NADPH has been proposed previously for P-450IIEl-catalyzed reactions (32). However, the magnitudes of k42, the rate of this hypothetical reaction, and kSl,the rate of dissociation of the EP complex, are not known. The molecular basis for the presently observed isotope effect remains to be investigated further. (k34
Acknowledgment. We thank Dr. Larry K. Keefer for providing [2H6]NDh4Afor this study, Drs. James Gillette and Jeong-Sook H. Yoo for valuable discussions, Ms.Fang Xiao for assistance in the preparation of microsomes, and
Ms. Dorothy Wong for excellent secretarial assistance. Redst4 NO.NDMA, 62-75-9;DMF,68-12-2;Dz, 7782-39-0; acetone, 67-64-1; ethanol, 64-17-5; cytochrome P-450,9035-51-2; NDEA deethylase, 127121-61-3;p-nitrophenol hydroxylase, 91116-87-9;NDMA demethylase, 69772-95-8.
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Enzymatic Oxidation of Ethyl Carbamate to Vinyl Carbamate and Its Role as an Intermediate in the Formation of 1,N'-Ethenoadenosine F. Peter Guengerich* and Dong-Hyun Kim? Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received December 21, 1990 The carcinogen ethyl carbamate has been postulated to be activated by oxidation to vinyl carbamate and then to an epoxide which can react with nucleic acids [Dahl, G. A., Miller, J. A., and Miller, E. C. (1978)Cancer Res. 38,3793-38041. To date, the enzymatic conversion of ethyl carbamate to vinyl carbamate had not been demonstrated. Recently, we obtained evidence that the same cytochrome P-450enzyme (P-4502E1) is involved in the oxidation of both ethyl carbamate and vinyl carbamate [Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991)Chen. Res. Toxicol. 4, 168-1791, When human liver microsomes were incubated with NADPH and ethyl carbamate, the products vinyl carbamate, 2-hydroxyethyl carbamate, and ethyl Nhydroxycarbamate were detected by use of (a) combined capillary gas chromatography/chemical ionization mass spectrometry or (b) high-performance liquid chromatography of radioactive materials. A K, of -54 r M was estimated for the conversion of vinyl carbamate to 1,Methenoadenosine (in the presence of adenosine), but when the reaction was done with ethyl carbamate as the substrate, the rate of product formation was nearly first order in ethyl carbamate concentration (K, > 2 mM) and the rate was considerably slower than in the case of vinyl carbamate. The model derived with these parameters predicts a steady-state level of 0.22 pM vinyl carbamate, consonant with the value of -0.2 pM estimated experimentally. A large kinetic deuterium isotope effect (>7)was observed for the formation of 1,I@-ethenoadenosine from ethyl carbamate, and high isotope effects (6-8)were also noted for the formation of vinyl carbamate and 2-hydroxyethyl carbamate. These results are consistent with the view that the enzyme cytochrome P-4502E1 abstracts a hydrogen atom from the terminal carbon of ethyl carbamate: subsequent oxygen rebound yields 2-hydroxyethyl carbamate, while abstraction of an electron/proton pair from the radical yields vinyl carbamate. The rapid oxidation of vinyl carbamate to its epoxide is then catalyzed by the same enzyme. Such a reaction may be expected to be more general for ethyl compounds containing good leaving groups, and 1,M-ethenoadenosine was formed from ethyl bromide. Ethyl carbamate (urethan) has long been known to be carcinogenic at multiple sites in rodents, particularly in the lung (I). This cancer suspect is encountered in the chemical and tanning industries and-potentially of even greater concern-is present at low levels in fermented foods and beverages from the reaction of ethanol with carbamyl Present address: Korea Institute of Science and Technology, P.O. Box 131, Chongyangni, Seoul, South Korea.
phosphate. A striking structure-activity relationship is seen for tumorigenesis-neither methyl nor propyl carbamate is nearly as carcinogenic (2). The chemistry involved in the activation of ethyl carbamate t~ its ultimate carcinogenic form has been difficult to elucidate. Ethyl carbamate is not mutagenic itself, even in the presence of a microsomal activation system. A known metabolite, ethyl N-hydroxycarbamate,was viewed as a reactive form in the earlier literature (2),but it is now
0 1991 American Chemical Society OS93-228~/91/2704-0413~02.50/0
