Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.     

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.     

Cytochrome P-450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters

Cytochrome P-450 (P-450)-catalyzed oxidation of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester gives rise to 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester and to 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester, identified in this work. A pyridine hydroxymethyl diester of the sort of the latter compound is novel; under acidic or dehydrating conditions the diester is readily converted to a cyclic lactone (2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid 5-ethyl ester lactone). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester was not hydroxylated to form this hydroxymethyl compound or lactone, but 1,4-dihydro-2-hydroxymethyl-4-phenyl-6-methyl-3,5-pyridinedicarboxyli c acid diethyl ester was enzymatically oxidized to give both products. The rates of oxidative carboxylic ester cleavage and methyl hydroxylation varied among individual forms of P-450 tested. Experiments with 2H and 3H labels were used to estimate an intrinsic kinetic deuterium isotope effect of 15 for ethyl ester cleavage by rat liver P-450PB-B in a reconstituted system. Rat liver microsomal systems showed kinetic deuterium and tritium isotope effects of 8 and 11, respectively, and this deuterium isotope effect was not attenuated in either intra- or intermolecular competitive experiments. When deuterium was present in the ethyl (ester) groups, increases in the rate of 2-methyl hydroxylation were observed in rat liver microsomes and with purified P-450 beta NF-B (but not with P-450PB-B). Deuteration of the methyl groups gave rise to kinetic isotope effects of 7-11, but no increases were seen in the rates of ester cleavage. These studies and those on rates of substrate disappearance indicate that isotopically sensitive branching (metabolic switching) observed in these systems is not necessarily bidirectional.     

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.     

Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k(on) approximately 10(6) m(-1) s(-1)), and the k(off) rates range from 5.7 to 36 s(-1) (at 23 degrees C). Reduction of ferric P450 2A6 is rapid (7.5 s(-1)) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k > or = 10(6) m(-1) s(-1)), and the putative Fe2+.O2 complex decayed at a rate of approximately 0.3 s(-1) at 23 degrees C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b5. Kinetic analyses showed that approximately 1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6-10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s.     

Cytochrome P450 7A1 cholesterol 7alpha-hydroxylation: individual reaction steps in the catalytic cycle and rate-limiting ferric iron reduction

Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent K(d) of 0.51 μM, (ii) a rapid reduction rate in the presence of cholesterol (～10 s(-1) for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O(2) complex (29 s(-1)), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H(2)O and H(2)O(2) formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.     

Oxidation of methoxyphenethylamines by cytochrome P450 2D6. Analysis of rate-limiting steps

Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction ( approximately 30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3- and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3- or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular non-competitive deuterium isotope effects of 3.1-3.8 were measured for k(cat) and k(cat)/K(m) for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe(2+)O(2)(-) protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k(cat), and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions.     

Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450

Phenylacetylene and biphenylacetylene are oxidized by cytochrome P-450 to the corresponding arylacetic acids. The acetylenic hydrogen shifts to the adjacent carbon and one atom of molecular oxygen is incorporated into the carboxylic acid group in these transformations, which are subject to a large kinetic isotope effect when the acetylenic hydrogen is replaced by deuterium. The same products and isotope effects are observed when the two arylacetylenes are oxidized by m-chloroperbenzoic acid rather than by the enzyme. In contrast, the inactivation of cytochrome P-450 that occurs during the oxidation of phenylacetylene is insensitive to deuterium substitution. The partition ratio between metabolite formation and enzyme inactivation consequently changes from 26 to 15 in going from phenylacetylene to the deuterated analogue. Metabolite formation therefore diverges from heme alkylation very early in the catalytic process.     

Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.     

Cytochrome P450 3A4-catalyzed testosterone 6beta-hydroxylation stereochemistry, kinetic deuterium isotope effects, and rate-limiting steps

Testosterone 6beta-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6beta-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6beta-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-(2)H(5); (ii) 6,6-(2)H(2); (iii) 6alpha-(2)H; (iv) 6beta-(2)H; and (v) 6beta-(3)H testosterone. Only the 6beta-hydrogen was removed by P450 3A4 and not the 6alpha, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the beta face. Analysis of the rates of hydroxylation of 6beta-(1)H-, 6beta-(2)H-, and 6beta-(3)H-labeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ((D)k) of 15. The deuterium isotope effects on k(cat) and k(cat)/K(m) in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6beta-(2)H substitution. The high (D)k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high (D)k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high (D)k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.     

Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes

Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.     

Rat liver microsomal enzyme catalyzed oxidation of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine.

As part of our ongoing studies to characterize the catalytic pathway(s) for the monoamine oxidase and cytochrome P450 catalyzed oxidations of 1,4-disubstituted 1,2,3,6-tetrahydropyridinyl derivatives, we have examined the metabolic fate of 4-phenyl-trans-1-(2-phenylcyclopropyl)-1,2,3,6-tetrahydropyridine in NADPH supplemented rat liver microsomes. Three metabolic pathways have been identified: (1) allylic ring alpha-carbon oxidation to yield the dihydropyridinium species, (2) nitrogen oxidation to yield the N-oxide and (3) N-dealkylation to yield 4-phenyl-1,2,3,6-tetrahydropyridine and cinnamaldehyde. A possible mechanism to account for the formation of cinnamaldehye involves an initial single electron transfer from the nitrogen lone pair to the iron oxo system Fe(+3)(O) to form the corresponding cyclopropylaminyl radical cation that will be processed further to the final products. The reaction pathway leading to the dihydropyridinium metabolite may also proceed via the same radical cation intermediate but direct experimental evidence to this effect remains to be obtained.     

Deuterium isotope effect measurements on the interactions of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine with monoamine oxidase B

Kinetic deuterium isotope effects for the noncompetitive, intermolecular monoamine oxidase B-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the corresponding 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+ were found to be 3.55 on Vmax and 8.01 on Vmax/Km with MPTP-6,6-d2 as the deuterated substrate. Similar values were obtained with MPTP-2,2,6-d4 and MPTP-CD3-2,2,6,6-d4. The deuterium isotope effect for the electrochemical oxidation of 1 mM MPTP-2,2,6,6-d4 was only 1.35. These results indicate that the monoamine oxidase B-catalyzed oxidation of this substrate may not proceed via a reaction pathway involving alpha-carbon deprotonation of an aminium radical intermediate. Isotope effect measurements also established that the rate of inactivation of monoamine oxidase B by MPTP is unaffected by replacement of the C-6 methylene protons with deuterons, but is retarded by replacement of the C-2 methylene protons (DKi = 1.9). The mechanism-based inactivation of monoamine oxidase B by MPTP, therefore, is likely to mediated by a species derived from the enzyme-generated 2,3-dihydropyridinium oxidation product.     

Use of isotope effects to characterize intermediates in mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

A mechanism for beta-chlorophenethylamine inhibition of dopamine beta-monooxygenase has been postulated in which bound alpha-aminoacetophenone is generated followed by an intramolecular redox reaction to yield a ketone-derived radical cation as the inhibitory species (Mangold, J.B., and Klinman, J.P. (1984) J. Biol. Chem. 259, 7772-7779). Based on the assumption that the ketone radical is the inhibitory intermediate, an analogous system was predicted and verified (Bossard, M.J., and Klinman, J.P. (1986) J. Biol. Chem. 261, 16421-16427). In the present study, the role of alpha-aminoacetophenone as the proposed intermediate in the inactivation by beta-chlorophenethylamine was examined in greater detail. From the interdependence of tyramine and alpha-aminoacetophenone concentrations, ketone inactivation is concluded to occur at the substrate site as opposed to potential binding at the reductant-binding site. Using beta-[2-1H]- and beta-[2-2H]chlorophenethylamine, the magnitude of the deuterium isotope effect on inactivation under second-order conditions has been found to be identical to that observed under catalytic turnover, D(kappa inact/Ki) = D(kappa cat/Km) = 6-7. By contrast, the isotope effect on inactivation under conditions of substrate and oxygen saturation, D kappa inact = 2, is 3-fold smaller than that seen on catalytic turnover, D kappa cat = 6. This reduced isotope effect for inactivation is attributed to a normal isotope effect on substrate hydroxylation followed by an inverse isotope effect on the partitioning of the enol of alpha-aminoacetophenone between oxidation to a radical cation versus protonation to regenerate ketone. These findings are unusual in that two isotopically sensitive steps are present in the inactivation pathway whereas only one is observable in turnover.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Kinetic isotope effects and 'metabolic switching' in cytochrome P450-catalyzed reactions

The mechanistic significance of a kinetic isotope effect on a cytochrome P-450catalyzed reaction depends, fundamentally, on the nature of the interaction of the substrate with the active site of the enzyme as well as on the nature of the chemistry of the reaction catalyzed. Consequently, kinetic isotope effects can be used to extract information on the topology of the enzyme and the mechanism of substrate oxidation. Kinetic isotope effect studies are sometimes accompanied by ‘metabolic switching’ or a change in metabolic pathway, catalyzed either by the same enzyme or by a different enzyme. For the specific case where ‘metabolic switching’ gives rise to a change in regional specificity for the cytochrome P-450 catalyzed metabolism of a compound, this change can be explained in terms of the topological constraints on substrate binding imposed by the active site of the enzyme.     

Stereospecificity and kinetic mechanism of human prenylcysteine lyase,an unusual thioether oxidase

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, as well as the stability of the thioether bond, poses a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that degrades a variety of prenylcysteines. Prenylcysteine lyase is a FAD-dependent thioether oxidase that produces free cysteine, an isoprenoid aldehyde, and hydrogen peroxide as products of the reaction. Here we report initial studies of the kinetic mechanism and stereospecificity of this unusual enzyme. We utilized product and dead end inhibitors of prenylcysteine lyase to probe the kinetic mechanism of the multistep reaction. The results with these inhibitors, together with those of other experiments, suggest that the reaction catalyzed by prenylcysteine lyase proceeds through a sequential mechanism. The reaction catalyzed by the enzyme is stereospecific, in that the pro-S hydride of the farnesylcysteine is transferred to FAD to initiate the reaction. With (2R,1'S)-[1'-(2)H(1)]farnesylcysteine as a substrate, a primary deuterium isotope effect of 2 was observed on the steady state rate. However, the absence of an isotope effect on an observed pre-steady-state burst of hydrogen peroxide formation implicates a partially rate-determining proton transfer after a relatively fast C-H (C-D) bond cleavage step. Furthermore, no pre-steady-state burst of cysteine was observed. The finding that the rate of cysteine formation was within 2-fold of the steady-state k(cat) value indicates that cysteine production is one of the primary rate-limiting steps in the reaction. These results provide substantial new information on the catalytic mechanism of prenylcysteine lyase.