0090-9556/03/3112-1481–1498$7.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 31:1481–1498, 2003
`
`Vol. 31, No. 12
`1140/1106831
`Printed in U.S.A.
`
`THE USE OF DEUTERIUM ISOTOPE EFFECTS TO PROBE THE ACTIVE SITE
`PROPERTIES, MECHANISM OF CYTOCHROME P450-CATALYZED REACTIONS, AND
`MECHANISMS OF METABOLICALLY DEPENDENT TOXICITY
`
`SIDNEY D. NELSON AND WILLIAM F. TRAGER
`
`Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington
`
`(Received April 2, 2003; accepted July 1, 2003)
`
`This article is available online at http://dmd.aspetjournals.org
`
`ABSTRACT:
`
`Critical elements from studies that have led to our current under-
`standing of the factors that cause the observed primary deuterium
`isotope effect, (kH/kD)obs, of most enzymatically mediated reac-
`tions to be much smaller than the “true” or intrinsic primary deu-
`terium isotope effect, kH/kD, for the reaction are presented. This
`new understanding has provided a unique and powerful tool for
`probing the catalytic and active site properties of enzymes, partic-
`ularly the cytochromes P450 (P450). Examples are presented that
`
`illustrate how the technique has been used to determine kH/kD, and
`properties such as the catalytic nature of the reactive oxenoid
`intermediate, prochiral selectivity, the chemical and enzymatic
`mechanisms of cytochrome P450-catalyzed reactions, and the rel-
`ative active site size of different P450 isoforms. Examples are also
`presented of how deuterium isotope effects have been used to
`probe mechanisms of the formation of reactive metabolites that
`can cause toxic effects.
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`substrate concentrations. The disparity between kH/kD and (kH/kD)obs
`is a consequence of the multistep nature (substrate binding, debinding,
`product release, etc.) of the catalytic cycle of enzymatically mediated
`reactions. If deuterium isotope effects are to be successfully applied to
`such reactions, then the relationship between kH/kD and (kH/kD)obs
`must be known, since experiment can only yield (kH/kD)obs. Fortu-
`nately,
`the relationship between kH/kD and (kH/kD)obs has been
`largely clarified (Northrop, 1975, 1978, 1981a; Cleland, 1982); and
`how partially rate-limiting steps exclusive of the bond-breaking step
`can mask kH/kD by lowering the magnitude of (kH/kD)obs is now
`clearly understood.
`Even the simplest of enzymatic reactions is composed of three
`components (Northrop, 1981b). The first involves substrate combin-
`ing with enzyme to form an enzyme-substrate complex. Rate con-
`stants k12 and k21 govern the process. The second component is the
`catalytic component that transforms the enzyme-substrate complex
`into an enzyme-product complex with rate constant k23 (Reaction 1).
`In this simplest of cases the catalytic step is considered to be irre-
`versible. The third component is the product release step that entails
`dissociation of the complex to free enzyme and product with rate
`constant k31. Equations 1 to 3 define the kinetic expressions for the
`maximum velocity, V, the Michaelis constant, K, and the ratio of the
`two, V/K (Northrop, 1975, 1981b). In analyzing the kinetic
`
`Historically, the determination of deuterium isotope effects has
`been a powerful tool to help unravel the intricacies of carbon-hydro-
`gen bond cleavage and define the mechanism of specific chemical
`reactions. The intrinsic primary isotope effect, kH/kD, for a reaction is
`the magnitude of the isotope effect on the rate constant for the
`bond-breaking step (C–H versus C–D) of the reaction and is related to
`the symmetry of the transition state for that step. The larger the
`isotope effect, the more symmetrical the transition state, with the
`theoretical limit being 9 at 37°C in the absence of tunneling effects
`(Bell, 1974). The chemical events leading to the transition state and
`subsequent formation of products are the descriptors of a chemical
`mechanism. It is the relationship of kH/kD to transition state that
`provides mechanistic insight. Thus, kH/kD is the quantity that needs to
`be known, and for homogenous chemical reactions, the experimen-
`tally observed deuterium isotope effect, (kH/kD)obs, where (kH/kD)obs
`is defined as the ratio of a kinetic parameter such as Vmax or Vmax/Km
`obtained from a nondeuterated substrate to a deuterated substrate, is
`identical to kH/kD.
`This is generally not true of enzymatically mediated reactions and
`is the reason for the much less successful application of deuterium
`isotope effects to such reactions until the system was better under-
`stood. The experimentally observed isotope effect, (kH/kD)obs, was
`invariably found to be much smaller than kH/kD, the intrinsic isotope
`effect for that reaction, thereby obscuring both meaning and mecha-
`nism. Even more perplexing was the observation that (kH/kD)obs for
`the same enzymatic reaction could vary with different experimental
`designs. For example, (kH/kD)obs determined at saturating substrate
`concentrations could differ from values determined at decreasing
`
`Address correspondence to: William Trager, Department of Medicinal Chem-
`istry, University of Washington, 1959 NE Pacific Street, Health Sciences Bldg.,
`H-364C, Seattle, WA 98195-7631. E-mail: trager@u.washington.edu
`
`1481
`
`V ⫽
`
`K ⫽
`
`k23k31Et
`k23 ⫹ k31
`k31(k12 ⫹ k23)
`k12(k23 ⫹ k31)
`k12k23Et
`k21 ⫹ k23
`
`V/K ⫽
`
`(1)
`
`(2)
`
`(3)
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`

`DV ⫽ VH/VD ⫽
`
`k23H/k23D ⫹ k23H/k31
`1 ⫹ k23H/k31
`k23H/k23D ⫹ k23H/k21
`D(V/K) ⫽ (V/K)H/(V/K)D ⫽
`1 ⫹ k23H/k21
`DV and D(V/K) are short-form terms introduced by Northrop (1975)
`for VH/VD and (V/K)H/(V/K)D, respectively. If eqs. 4 and 5 are
`rewritten in general form as eqs. 6 and 7, as proposed by Northrop
`(1975), the factors
`
`(4)
`
`(5)
`
`(6)
`
`(7)
`
`1482
`NELSON AND TRAGER
`“commitment to catalysis,” is a measure of the enzyme-substrate
`the expression of
`parameters, Northrop (1975) demonstrates that
`complex tendency to proceed through the first irreversible step versus
`deuterium isotope effects on V and V/K should be the most revealing
`reversing to free enzyme and substrate. The value of D(V/K) [the
`and important as they depend on the fewest variables and on rate
`constants for different parts of the kinetic scheme. Since V is deter-
`value of (kH/kD)obs for V/K conditions] will only be close to Dk if the
`mined at saturating concentrations, the binding component is elimi-
`first irreversible step is also the catalytic step and the reverse step to
`nated and its value reflects the catalytic and product release compo-
`free enzyme and substrate is fast relative to the catalytic step. What
`nents. V/K, which is reflective of the kinetics at
`low substrate
`makes Northrop’s presentation of isotope effects in this way so
`concentration, is dependent on all rate constants up to and including
`powerful is that steady-state equations for much more complex en-
`the first irreversible step. In this case it is also the catalytic step.
`zyme mechanisms can all be reduced to this form. R and C simply
`Isotope effects on V and V/K for this example are described by eqs.
`become more complex expressions of a collection of rate constants. In
`4 and 5.
`more complicated schemes involving a greater number of reversible
`steps, an additional grouping of rate constants, Cr, termed the “com-
`mitment to reverse catalysis,” can be factored from both DV and
`D(V/K) experiments. Cr is a measure of the substrates tendency to
`return to E ⫹ S. In such schemes DV experiments now contain both
`R and Cr, while D(V/K) experiments will contain Cr and Cf, where Cf
`is a collection of rate constants termed the “commitment to forward
`catalysis” that measures the substrate’s tendency to move forward to
`E ⫹ P. Despite the greater complexity, the general forms of the
`equations still indicate that modification of (kH/kD)obs relative to
`kH/kD can be readily understood in the interplay of a collection of
`specific rate constants.
`The simplest formulation for a cytochrome P450 (P4501)-catalyzed
`reaction involves one extra step over the general scheme presented
`above. This is the single irreversible step prior to substrate transfor-
`mation in which the enzyme-substrate complex, ES, is irreversibly
`transformed into the substrate-bound active oxygenating perferryl
`oxene enzymatic species, EOS, which then proceeds to the enzyme-
`product complex, EP, with the rate constant k34 (the isotopically
`sensitive step) (Korzekwa et al., 1989) (Reaction 2). The kinetics of
`the system have been described (Northrop, 1978, 1981; Cleland,
`1982) and the following equations for the isotope effect derived. The
`term k34H(1/k23 ⫹ 1/k41) in
`k34H/k34D ⫹ k34H(1/k23 ⫹ 1/k41)
`1 ⫹ k34H(1/k23 ⫹ 1/k41)
`(V/K)H/(V/K)D ⫽ DV/K ⫽ 1
`(9)
`eq. 8 is termed the V ratio and is the factor that will increasingly mask
`the intrinsic isotope effect, k34H/k34D; the larger k34 is relative to k23
`and k41. So if product formation is faster than product release, the
`formation of the perferryl oxene species, the magnitude of the intrinsic
`isotope effect, will be suppressed; i.e., (kH/kD)obs will be smaller than
`kH/kD. Thus, the faster product formation is, the greater the suppres-
`sion of kH/kD. Equation 9 indicates that for V/K conditions [(DV/K)],
`no isotope effect should be observed, i.e., kH/kDobs must equal 1. This
`is because the first irreversible step in the scheme, conversion of ES
`to EOS, is not isotopically sensitive. Once EOS is formed, it is
`committed to continue on to product irrespective of whether or not
`substrate contains deuterium. Under V/K conditions, when an irre-
`versible step precedes the isotopically sensitive step, the decreased
`rate constant, k34D, generated by deuterium substitution, will be
`compensated for by an equal and opposite increase in the concentra-
`tion of EOSD, i.e., (EOSH)k34H ⫽ (EOSD)k34D. Thus, for cytochrome
`P450 reactions, deuterium isotope effects on V/K should in general
`never be observed (Korzekwa et al., 1989). Isotope effects can only be
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`VH/VD ⫽ DV ⫽
`
`(8)
`
`DV ⫽
`
`D(V/K) ⫽
`
`Dk ⫹ R
`1 ⫹ R
`Dk ⫹ C
`1 ⫹ C
`that can lead to the observed isotope effect, DV or D(V/K), having a
`much lower value than the intrinsic isotope effect, Dk (Northrop’s
`nomenclature), become clear. In the equation for the isotope effect on
`V, R, termed the “ratio of catalysis,” is a measure of rate of the
`catalytic step relative to the rate of the other forward steps contribut-
`ing to maximal velocity. In the present example, the other forward
`step would be the product release step. The value of DV [the value of
`(kH/kD)obs for Vmax conditions] will only be close to Dk (intrinsic
`isotope effect, kH/kD) when product release is fast relative to the
`catalytic step. In the equation for the isotope effect on V/K, C, termed
`
`REACTION 1.
`
`REACTION 2.
`
`REACTION 3.
`
`1 Abbreviations used are: P450, cytochrome P450; NDMA, N-nitrosodimeth-
`ylamine; EDB, ethylene dibromide; GSH, glutathione; Tris-BP, tris(2,3-dibro-
`mopropyl)phosphate; DBCP, 1,2-dibromo-3-chloropropane; NMF, N-methylfor-
`mamide; NDPS, N-(3,5-dichlorophenyl)succinimide; 3-MC, 3-methylcholan-
`threne; BHT, butylated hydroxytoluene; VPA, valproic acid; 3MI, 3-methylindole.
`
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`USE OF DEUTERIUM ISOTOPE EFFECTS TO PROBE P450 REACTIONS
`
`1483
`
`FIG. 1. The (kH/kD)obs value for the O-demethylation of p-methoxyanisole determined from an experiment of intermolecular design (a) and intramolecular design (b).
`
`observed if some mechanism or some special experimental condition
`exists that diminishes the increase in EOSD that occurs in response to
`the decreased value of k34D relative to k34H. Fortunately, there are
`both mechanisms and experimental conditions that will tend to equal-
`ize (EOSH) and (EOSD) and thereby unmask the intrinsic isotope
`effect, i.e., cause (kH/kD)obs to approach kH/kD. One mechanism that
`can achieve this goal is the presence of a branched reaction pathway
`from the substrate-bound active oxygenating perferryl oxene species,
`EOSD, to a nonisotopically sensitive alternate product and free en-
`zyme. A second and similar mechanism is reduction of the perferryl
`oxene EOSD to free substrate, free enzyme, and water (Atkins and
`Sligar, 1987, 1988; Korzekwa et al., 1989). A special experimental
`condition that can achieve the same end is an isotope effect experi-
`ment of symmetrical intramolecular design (Hjelmeland et al., 1977;
`Miwa et al., 1980; Gelb et al., 1982; Lindsay Smith et al., 1984).
`Symmetrical Intramolecular Design
`In an isotope effect experiment of intramolecular design, a substrate
`is chosen that is susceptible to enzymatic attack at either of two
`symmetrically equivalent sites (Hjelmeland et al., 1977; Miwa et al.,
`1980; Gelb et al., 1982; Lindsay Smith et al., 1984). One site contains
`deuterium and the other retains its normal complement of hydrogen.
`The enzyme then has the choice of attacking a deuterated or an
`
`REACTION 4.
`
`equivalent protio site within the same molecule. It can be viewed as
`a special case of branching and can be modeled as shown. The
`observed isotope effect, (kH/kD)obs, measured as the ratio of product
`resulting from attack at the protio site versus product resulting from
`attack at the deutero site, PH/PD, reflects the intramolecular competi-
`tion between these two sites, i.e., PH/PD ⫽ kH[EOSH]/kD[EOSD]
`(Reaction 3). Kinetically, the experimental conditions correspond to a
`V/K isotope effect (Korzekwa et al., 1989). Thus, (kH/kD)obs is
`independent of all kinetic steps following branching of EOS and can
`be described by eq. 10. The equation reveals that the faster the rate of
`reorientation of the substrate is in the active site of the enzyme, k43,
`relative to the
`
`(10)
`
`PH/PD ⫽ (kH/kD)obs ⫽
`
`k45H/k45D ⫹ k45H/k43
`1 ⫹ k45H/k43
`commitment to catalysis (catalytic step), k45H, i.e., k43 ⬎ k45H, the more
`the concentrations of EOSD and EOSH will equalize and the closer
`(kH/kD)obs will be to k45H/k45D. Conversely, the higher the commitment
`to catalysis relative to the rate of reorientation, the more the intrinsic
`isotope effect will be suppressed. An early, if the not first, example of the
`power of an intramolecular isotope effect experiment to unmask the
`intrinsic isotope effect is provided by the work of Foster et al. (1974).
`These investigators measured the deuterium isotope effect associated
`with the oxidative O-demethylation of p-methoxyanisole using two dif-
`ferent experimental designs. The first was the traditional experiment of
`intermolecular design in which the rate of demethylation of the hydrogen-
`containing substrate was measured and compared with the rate of de-
`methylation of the deuterium-containing substrate determined in a sepa-
`rate experiment (Fig. 1a). The observed isotope, (kH/kD)obs, was found to
`be 2. The second experiment was of intramolecular design in which the
`hydrogens of one of the O-methyl groups were replaced with deuterium.
`Incubation of the substrate with a microsomal preparation of cytochrome
`P450 followed by measurement of the p-methoxyphenol to p-trideutero-
`methoxyphenol product ratio gave a (kH/kD)obs of 10 (Fig. 1b). The
`observed products, p-methoxyphenol and formaldehyde, are consistent
`with at least two distinct mechanisms that might be operative. The first
`would be an insertion mechanism in which an oxygen atom inserts
`between a carbon-hydrogen bond of one of the methoxy groups to
`generate a hemiacetal intermediate. The hemiacetal is then hydrolyzed to
`generate the observed products. This mechanism should be accompanied
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`1484
`
`NELSON AND TRAGER
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`FIG. 2. The (kH/kD)obs values for the formation of n-octanol from n-octane-1,8-2H2, n-octane-1-2H3, and n-octane-1,2,3-2H7.
`
`by a primary deuterium isotope effect of low magnitude, approximately
`2, since the transition state for oxygen insertion would necessarily be
`nonlinear (Shea et al., 1983). The second mechanism would involve
`initial hydrogen atom abstraction from the methyl group to form meth-
`ylene and hydroxy radicals followed by recombination to generate the
`intermediate hemiacetal. This is the mechanism first postulated by
`Groves et al. (1978) to account for the cytochrome P450-catalyzed
`hydroxylation of norbornane, a mechanism that is now generally ac-
`cepted as the mechanism for all cytochrome P450-catalyzed hydroxyla-
`tions of aliphatic carbon-hydrogen bonds. Since direct hydrogen abstrac-
`
`tion would, if possible, involve a linear transition state, the magnitude of
`the primary deuterium isotope effect could approach the theoretical value
`of approximately 9, depending upon the symmetry of the transition state.
`The (kH/kD)obs of 2 given by the intermolecular isotope effect experi-
`ment is supportive of a mechanism involving oxygen insertion. In con-
`trast, the (kH/kD)obs of 10 from the intramolecular isotope effect exper-
`iment is only consistent with the abstraction recombination mechanism.
`Since (kH/kD)obs from the intramolecular isotope effect experiment is
`much closer to kH/kD than is (kH/kD)obs from the intermolecular exper-
`iment, the choice between possible mechanisms is clear. Indeed the
`
`FIG. 3. The intrinsic isotope effect, kH/kD, for the cytochrome P450-catalyzed hydroxylation of (1,2)-dideuteromethyl-o-xylene (a) and (1,2*)-dideuteromethyl-p-xylene
`(b).
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`1485
`
`(11)
`
`(kH/kD)obs ⫽
`
`USE OF DEUTERIUM ISOTOPE EFFECTS TO PROBE P450 REACTIONS
`k45H/k45D ⫹ k45H/(k43 ⫹ k46)
`1 ⫹ k45H/(k43 ⫹ k46)
`The values of (kH/kD)obs found for cytochrome CYP2B1-catalyzed
`␻-hydroxylation of the dideuterated analog of n-octane, n-octane-1,8-
`2H2, the heptadeuterated analog, n-octane-1,2,3-2H7, and the trideu-
`terated analog, n-octane-1-2H3, of 16.11, 4.0, and 11.77, respectively,
`nicely illustrate the effects of k43 and k46 (Fig. 2). Before comparing
`values, it is first necessary to determine kH/kD for the reaction. Of the
`three substrates, the ␻-hydroxylation of n-octane-1,8-2H2 can best be
`expected to provide kH/kD. Both terminal methyl groups are isotopi-
`cally equivalent, and it can be assumed that the inherent rate of
`rotation of a methyl group is a much faster process than the rate of
`cleavage of a carbon-hydrogen bond via hydrogen atom abstraction.
`Thus, the enzyme always has the choice of oxidizing a carbon-
`hydrogen or carbon-deuterium bond irrespective of which methyl
`group is oriented for catalysis. The value of (kH/kD)obs ⫽ 16.11 for
`this substrate corresponds to an intrinsic primary isotope effect of 9.18
`once it has been corrected for the contribution of secondary isotope
`effects and the fact that the methyl group contains two hydrogens but
`only one deuterium (Jones and Trager, 1987). As indicated above, an
`intrinsic primary isotope effect of 9 is the theoretical limit for cleav-
`age of a carbon-hydrogen bond in the absence of tunneling effects
`(Bell, 1974). This provides compelling evidence that the cytochrome
`CYP2B1-catalyzed ␻-hydroxylation of n-octane involves a highly
`symmetrical transition state and is consistent with the abstraction-
`recombination mechanism for aliphatic hydroxylation (Groves et al.,
`1978). In the case of n-octane-1,2,3-2H7, the enzyme has the choice of
`oxidizing either a carbon-hydrogen bond or a carbon-deuterium bond,
`depending on which terminal methyl group of the substrate is properly
`oriented for catalysis. According to eq. 11, if (kH/kD)obs is to be close
`to kH/kD, the rate of methyl group interchange, k43, must be much
`faster than the rate of bond breaking, k45H. The (kH/kD)obs of 4
`indicates that this is clearly not true for this substrate and suggests that
`the distance between terminal methyl groups is large enough so that
`the rate of interchange is slow enough to prevent the concentrations of
`[EOSH] and [EOSD] from equalizing. For the case of n-octane-1-2H3,
`the enzyme also has the choice of oxidizing either a carbon-hydrogen
`bond or a carbon-deuterium bond, depending on which terminal
`methyl group of the substrate is properly oriented for catalysis. What
`is different about this substrate relative to n-octane-1,2,3-2H7 is that it
`also has the choice of a branched reaction pathway, (␻-1)-hydroxy-
`lation, that is blocked by deuteration in the case of n-octane-1,2,3-2H7.
`
`FIG. 4. The cytochrome P450-catalyzed N-demethylation of various (X ⫽ H, Cl,
`CN, and NO2) p-substituted N,N-dimethylaniline analogs.
`(kH/kD)obs of 10, which is a composite value for one primary and two
`secondary isotope effects, i.e., PS2 (Hanzlik et al., 1985) is still large
`enough to suggest that the transition state is close to symmetrical and that
`the primary isotope effect is not too far from its maximum value.
`Although the ratio of the concentration of [EOSH] to the concen-
`tration of [EOSD] must equal 1 for (kH/kD)obs to be equal to kH/kD,
`rapid equilibration of equivalent but isotopically distinct intramolec-
`ular oxidation sites is not the only means by which this condition can
`be met. As indicated above, the presence of a branched reaction
`pathway from the substrate-bound active oxygenating perferryl oxene
`species, EOSD, to a nonisotopically sensitive alternate product and
`free enzyme or reduction of the perferryl oxene EOSD to free sub-
`strate, free enzyme, and water can also equalize [EOSH] and [EOSD]
`and lead to (kH/kD)obs being equal to kH/kD. A model for a branched
`reaction pathway in competition with the isotopically sensitive step
`follows (Harada et al., 1984; Jones et al., 1986). In the model,
`substrate can reversibly reorient in the active site of the enzyme with
`rate constants k34 and k43 (Reaction 4). This allows it to present either
`the deutero site, EOSD, or the protio site, EOSH, for catalysis. EOSH
`and EOSD then go on to form products. P1 is formed from EOSH and
`EOSD with rate constants, k45H or k45D, respectively, whereas a single
`rate constant, k46, characterizes the formation of P2 from EOSH and
`EOSD. This is because P2 arises from oxidative attack at a molecular
`site remote from the site of deuterium substitution. The observed
`isotope effect for the model is given by eq. 11. Equation 11 reveals
`that the rates of substrate reorientation in the active site, k43, and
`formation of P2, k46, relative to the rate of formation of isotopically
`sensitive P1 are the factors that govern the magnitude of (kH/kD)obs
`and define the degree of masking of k45H/k45D. The larger k43 and/or
`k46 are relative to k45H, the closer (kH/kD)obs will be to k45H/k45D.
`
`FIG. 5. The fixed intramolecular distance between methyl and trideuteromethyl groups of o-xylene-␣-2H3, p-xylene-␣-2H3, 2-2H3-6-dimethylnaphthalene, and 4-2H3-4⬘-
`dimethylbiphenyl.
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`1486
`
`REACTION 5.
`
`FIG. 6. The cytochrome P450-catalyzed formation of (S)- and (R)-2-methyl-2-
`phenylethanol.
`
`TABLE 1
`Value of the isotope effect expected for the cytochrome P450 hydroxylation of a
`substrate at a nondeuterated site by parallel pathway, nondissociative, and
`dissociative mechanisms determined from competitive and noncompetitive
`experiments
`
`NELSON AND TRAGER
`carbon-hydrogen bond. This is because the magnitude of kH/kD varies
`as a bell-shaped curve and correlates with transition state symmetry.
`The magnitude of kH/kD is maximum when the transition state is most
`symmetrical and declines when the transition state is either more
`reactant-like or more product-like (Melander, 1960; Westheimer,
`1961; Hammond, 1965). It is therefore not surprising that the intrinsic
`primary isotope effect associated with the benzylic hydroxylation of
`o- and p-xylene by various microsomal preparations and purified
`P450s is less than maximal, falling within the range of 5.2 to 7.4 (Fig.
`3) (Hanzlik and Ling, 1993; Iyer et al., 1997). Because the benzylic
`radical can be resonance stabilized, the less than maximal value for
`the intrinsic isotope effect for benzylic hydroxylation of xylene indi-
`cates that the transition state for the reaction is more reactant-like, and
`the value of the isotope effect lies on the ascending portion of the
`bell-shaped curve.
`Another factor, besides substrate structure, that could affect the
`symmetry of the transition state for hydrogen atom abstraction is the
`active site structure of the enzyme itself. This raises the question of
`whether or not different P450s can catalyze the same oxidative reac-
`tion with different kH/kD values. If they can, it must mean that either
`different P450s can catalyze the same reaction with different mecha-
`nisms or the symmetry of the transition state can vary as a function of
`different active site topographies by modulating the stability of the
`activated oxygen-substrate complex. The activated oxygen atom of
`cytochromes P450 is an extremely reactive species that is capable of
`oxidizing virtually any carbon-hydrogen bond with which it comes in
`contact. These enzymes are different from most other enzymes in that
`the energy cost for catalysis is in generating the active oxygen species
`rather than in orienting the substrate toward a transition state-like
`structure by specific binding. This suggests that the mechanism of a
`given reaction catalyzed by different P450s is likely to be unchanged.
`In this regard, considerable evidence exists indicating that aliphatic
`hydroxylation reactions, irrespective of enzyme, proceed by a hydro-
`gen atom abstraction-recombination mechanism. Thus, if differences
`are found, they are likely to be caused by differences in active site
`topographies. To probe this question, the isotope effects for the
`␻-hydroxylation of n-octane by three very different P450s, CYP1A1,
`CYP2B1, and CYP2B4, were determined (Jones et al., 1990). All
`three enzymes gave virtually identical intrinsic primary isotope effects
`of approximately 9, even though the ␻- to (␻-1)-hydroxylation prod-
`uct ratio was very different for each isoform and the individual P450s
`were from different species (rat and rabbit) and different enzyme
`families. The N-demethylation of a series of para substituted (H, Cl,
`CN, NO2) dimethylanilines (Fig. 4) catalyzed by CYP1A2, CYP2B1,
`CYP4B1, and CYP101 gave similar findings (Karki et al., 1995).
`Whereas the magnitude of the intrinsic isotope effect varied from
`substrate to substrate, it was virtually identical across enzymes. The
`variation in the magnitude of the intrinsic isotope effect between
`substrates is expected because the transition state for the N-demeth-
`ylation of this series of substrates would fall on the ascending slope of
`the bell-shaped curve that defines the relationship between transition
`state and the magnitude of the isotope effect. Different aromatic
`substituents would differ in their ability to stabilize the radical inter-
`mediate. The picture that emerges from these data is that any given
`cytochrome P450-catalyzed reaction is likely to proceed by the same
`mechanism with the same value for the intrinsic isotope effect, irre-
`spective of the isoform catalyzing the reaction.
`The lack of sensitivity to individual P450s and different active
`site architectures of the intrinsic primary isotope effect for ali-
`phatic hydroxylation suggests that the masking effect might be
`used to explore effective active site dimensions. As has been
`indicated above, the concentrations of [EOSH] and [EOSD] for
`
`Isotope Effect
`
`Competitive VH/VD
`Noncompetitive
`(V/K)H/(V/K)D
`
`Parallel
`Pathway
`1.0
`1.0
`
`Mechanism
`
`Nondissociative
`⬍1.0
`⬍1.0
`
`Dissociative
`1.0
`⬍1.0 to
`⬎1.0
`
`Although both substrates will form the (␻-1)-product, it will form at
`a much faster rate when n-octane-1-2H3 is the substrate. The value of
`11.77 for (kH/kD)obs for n-octane-1-2H3 corresponds to a primary
`isotope effect of 9.14, when the secondary isotope effect contribution
`is removed, indicating that k46 is much larger than k45H, so that the
`sum (k43 ⫹ k46) is now big enough to overwhelm k45H and allow full
`expression of the intrinsic isotope effect. The dominance of k46 over
`k45H is substantiated by the product ratio. 2-Octanol is formed 23
`times faster than 1-octanol.
`Establishing that the ␻-hydroxylation of n-octane proceeds with a
`maximum intrinsic isotope effect of 9, and therefore a highly sym-
`metrical transition state, provides an important limit for the mecha-
`nistic interpretation of all cytochrome P450 reactions that involve
`cleavage of a carbon-hydrogen bond. It means that cleavage of a
`carbon-hydrogen bond of any saturated carbon atom having a differ-
`ent substitution pattern (secondary, tertiary, substituted with other
`atoms in addition to hydrogen) must proceed with a lower intrinsic
`isotope effect and a less symmetrical transition state. The degree to
`which the intrinsic isotope effect departs from maximum and the
`transition state from symmetrical depends on the difference that that
`specific substitution pattern engenders relative to a primary aliphatic
`
`Auspex Exhibit 2006
`Apotex v. Auspex
`IPR2021-01507
`Page 6
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`USE OF DEUTERIUM ISOTOPE EFFECTS TO PROBE P450 REACTIONS
`
`1487
`
`FIG. 7. The CYP2C11-catalyzed hydroxylation of testosterone to 2␣-hydroxytestosterone and 16␣-hydroxytestosterone.
`
`that F87 in the wild-type enzyme occupies active site space that
`effectively leads to a smaller active site that is more restrictive
`toward larger substrates (Rock et al., 2002).
`Nonsymmetrical Intramolecular Design
`More than a single product can sometimes be formed by the action
`of a single P450 on a single substrate. If one of the intramolecular
`product-forming sites is deuterated, the possibility of formation of an
`alternate product from a different site could be in competition with the
`deuterated site and thereby modulate the value of (kH/kD)obs. A
`model describing this possibility is shown below (Korzekwa et al.,
`1989, 1995). The model assumes that the substrate is free to reorient
`within the enzyme-substrate complex, EOS, rapidly, relative to the
`rate of substrate oxidation, k34 and k35 (Reaction 5). When P1 is the
`isotopically sensitive step while P2 is in competition with P1 but is not
`itself isotopically sensitive, DVP1 and D(V/K)P1 are given by eqs. 12
`and 13, respectively. It is apparent from the model that an isotope
`effect on k34, k34D, will alter the ratio of products, P1 and P2.
`Equation 12 also indicates that the larger k35 is relative to k34, the
`closer (kH/kD)obs will approach the intrinsic isotope effect, k34H/k34D.
`Furthermore, if product release from the isotopically sensitive path-
`way, k41, is slow, k34H/k34D will tend to be masked. In contrast, if
`product release from the alternate pathway, k51, is slow, k34H/k34D
`will tend to be unmasked.
`
`isotopically nonequivalent but otherwise identical catalytic sites
`within a substrate must be equal for (kH/kD)obs to be equal to
`kH/kD. The larger and less impeded an active site and/or the
`smaller the intramolecular distance between protium and deute-
`rium sites, the less likely the catalytic event will occur before
`equilibration of [EOSH] and [EOSD]. A set of substrates for ali-
`phatic hydroxylation with a range of known fixed distances be-
`tween protium and deuterium sites might be expected to display a
`range of (kH/kD)obs values for different P450 isoforms reflecting
`different active site topographies. The concept was tested and
`verified with CYP101, a P450 with a known active site, using the
`benzylic hydroxylation of o- and p-xylene-␣-2H3 and 4-2H3,4⬘-
`dimethylbiphenyl, in which the protio and deuterio catalytic sites
`vary by 2.5 Å, 6.6 Å, and 11 Å, respectively (Audergon et al.,
`1999). Results from CYP2B1, several microsomal preparations,
`and the same set of substrates indicated that an intramolecular
`distance of less than 7 Å between catalytic sites allowed [EOSH]
`and [EOSD] to equalize prior to catalysis, whereas a distance
`greater than 11 Å led to complete suppression of kH/kD (Iyer et al.,
`1997). In a subsequent study, 2-2H3, 6-dimethylnaphthalene (Fig.
`5), was added to the set of additional fixed distance probes (methyl
`versus trideuteromethyl groups) to explore the active site topog-
`raphy of CYP4B1 relative to CYP2B1 and CYP102 (Henne et al.,
`2001). The results of the study led to the conclusion that the active
`site of CYP4B1 is considerably restricted relative to CYP2B1. In
`a similar study, p-xylene-␣-2H3 an