Quantitative Production of Compound I
A R T I C L E S
in Kbind results at -50 °C. It is noteworthy that, for the aromatic
substrate BA, extrapolation of Kbind to ambient temperature gives
a value that is about an order of magnitude smaller than that
for lauric acid.25 The CYP119 Compound I binding constants
are smaller than the binding constants for the same substrates
in the resting enzyme,36 but the order of substrate binding
appears to be the same.
If tunneling is significant, then the Arrhenius plot in Figure
5B would be the tangent to a curve with the calculated activation
energy smaller than the true value for the chemical reaction,37
and accordingly, the extrapolated apparent second-order rate
constant at ambient temperature would be smaller than the actual
value. Our view is that this rationalization is reasonable because
tunneling in BA oxidations by a model for Compound I, an
iron(IV)-oxo porphyrin radical cation, was considerable.28 In
addition, as discussed later, comparison of the kinetic isotope
effects at -50 and 22 °C also suggests a significant tunneling
component in the hydroxylation reaction at low temperature.
The first-order rate constants for oxidations of the benzyl
alcohol isotopomers are perhaps the most important findings in
this work. The bond dissociation energy (BDE) of the benzylic
C-H bonds in benzyl alcohol is relatively small, estimated to
be only 79 kcal/mol,38 but oxidation of this substrate by CYP119
Compound I is not phenomenally fast. In fact, CYP119
Compound I reactions with BA, lauric acid,25 benzphetamine,27
and a methylcyclopropyl group26 are about 2 orders of magni-
tude faster than reactions of Compound I models lacking the
thiolate ligand.39
It is noteworthy for analysis of P450 Compound I reactions,
in general, that the rate constants obtained for oxidations by
CYP119 Compound I are not a special feature of the particular
P450 enzyme. In two kinetic studies,26,27 rate constants for
oxidations by Compound I of the mammalian hepatic P450
enzyme CYP2B4 and those for CYP119 Compound I were
directly compared. For oxidations of benzphetamine and a
methylcyclopropane, the rate constants for oxidation by Com-
pound I from CYP2B4 were greater than those for oxidations
by Compound I of CYP119 by factors of 1.5 and 1.2,
respectively.26,27 The differences in activation free energies for
the two P450 Compounds I were small in these examples, ∆∆Gq
< 0.3 kcal/mol, and, in fact, smaller than the difference for the
rate constants for oxidation of benzphetamine by Compounds I
from CYP2B4 and its F429H mutant.27 In another kinetic study
that can be used for comparisons of Compounds I,40 rate
constants for oxidations by Compound I of the heme-thiolate
enzyme chloroperoxidase (CPO) from Caldariomyces fumago
were similar to those found for CYP119 Compound I. From all
kinetic data now available, Compound I from CYP119 appears
to be normal in its C-H oxidizing reactivity.
The type of kinetics and energetics information available from
the variable temperature studies of reactions of CYP119
Compound I was not available previously for any P450 enzyme,
and it represents important benchmark results for computational
studies. In that regard, it must be noted that the binding constants
for substrates in the activated CYP119 Compound I are not the
same as those for substrates binding in the resting CYP119
enzyme. In the case of lauric acid, for example, the binding
constant for resting CYP11936 is much larger than the binding
constant for CYP119 Compound I.25 Because experimentally
derived apparent second-order rate constants are the products
of the binding constant and the first-order rate constant (see
below for examples), one cannot predict rate constants for P450
Compound I oxidations without estimating both the binding
constant and activation energy values. Computations of the first-
order rate constants might give general results that apply for
various P450s, but binding constants for different P450s will
inevitably be highly variable and difficult to predict.
When the product of the binding constant and substrate
concentration is small, the denominator in eq 1 reduces to unity,
and a series of studies at varying concentration of substrate under
pseudo-first-order conditions give kobs values that are described
by a straight line. In such a case, eq 1 reduces to eq 4, where
the linear result is the tangent to the curve for saturation kinetic
results. The straight line in a plot described by eq 4 gives an
apparent second-order rate constant (kApp), which is the product
of the binding constant and the first-order oxidation rate constant.
In LFP studies of CYP119 Compound I at 22 °C, a linear
dependence on substrate concentration was found for BA
oxidation reactions,25 and one can compare those results to
results from this study extrapolated to 22 °C.
kobs - k0 ) Kbindkox[Subs] ) kApp[Subs]
(4)
Extrapolation of the BA-d2 results to ambient temperature is
shown in the plots in Figure 5. At 22 °C, the binding constant
is Kbind ) 48 M-1, and the rate constant for oxidation of BA-d2
is kox ) 160 s-1. These results predict that the apparent second-
order rate constant for BA-d2 oxidation at 22 °C will be 7.7 ×
The difference between the measured reactivities of Com-
pounds I of CYP119, CYP2B4, and CPO and the inferred
reactivities of the oxidizing transients in other P450 enzymes
is a contradiction, the resolution of which will require further
study. The inability to detect P450 Compound I from reactions
of resting enzyme with hydroperoxy compounds might be
rationalized as due to a fast oxidase-type reaction where
Compound I reacts with a second molecule of hydroperoxy
compound to give molecular oxygen and a second molecule of
reduced hydroperoxy compound. The failure to observe CYP101
in low temperature radiolytic reduction experiments,41-44
however, cannot be explained by a conventional interpretation.
103 M-1 s-1. In fact, the experimental value for oxidation of
25
perdeuterated BA was kApp ) 1.17 × 104 M-1 s-1
,
and the
predicted result from the present work represents a 34%
reduction in the rate constant from the measured apparent
second-order rate constant found in the LFP study at ambient
temperature. The origin of the difference in the kinetic values
is not known with certainty, and there are at least two possible
explanations.
It is possible, in principle, that the different buffer solutions
in the two studies resulted in slightly different binding constants.
In the present work, the buffer contained 50% glycerol, but no
glycerol was added to the buffer in the LFP study. Although
this rationalization cannot be excluded, we favor a second
possibility involving a considerable amount of tunneling at -50
°C.
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(40) Zhang, R.; Nagraj, N.; Lansakara, D. S. P.; Hager, L. P.; Newcomb,
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(41) Davydov, R.; Macdonald, I. D. G.; Makris, T. M.; Sligar, S. G.;
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(42) Davydov, R.; Makris, T. M.; Kofman, V.; Werst, D. E.; Sligar, S. G.;
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