1
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M.J. Traylor et al. / Archives of Biochemistry and Biophysics 505 (2011) 186–193
and pH (Fig. 5b) indicating that coupling is altered by these vari-
ables. To the best of our knowledge this is the first investigation
of P450 coupling as a function of buffer type, pH, or temperature.
However Schenkman et al. observed an increase in the coupling
of the benzphetamine N-demethylation activity of CYP2B4 en-
riched rabbit liver microsomes with increasing phosphate buffer
concentration [22]. This observation contrasts with our own data
where buffer concentration did not alter coupling, suggesting that
P450 coupling may be differentially regulated by reaction condi-
tions for different P450 systems.
circular dichroism [39]. It is interesting that this change in struc-
ture could accompany a large change in activity without also per-
turbing the active site and altering the substrate binding
interactions relevant to selectivity. Schrag et al., however, observed
constant selectivity in the presence of a structural perturbation of
the active site in the CYP3A4 metabolism of triazolam. Regioselec-
tivity was unaffected by a change in active site topography induced
by magnesium ions [17]. Overall, it is clear that more work is
needed to understand the complex interactions between environ-
mental parameters and P450 function.
Similarly, regioselectivity was also not significantly altered by
temperature or phosphate buffer concentration (Fig. 5c). It was
found that 21.7% of the oxidized EOMCC was used to generate
the O-dealkylation product 7HCC in pH 6.7 phosphate solution.
While no previous study has investigated the effect of temperature
on regioselectivity, similar trends with buffer concentration have
been observed [14,16]. Buffer concentration did not alter the ratio
of several testosterone metabolites from purified rat CYP2A1,
CYP2B1, and CYP2C11 [14] or of midazolam metabolites from hu-
man liver microsomes [16], consistent with our findings that regi-
oselectivity was insensitive to buffer concentration. However, a
change in regioselectivity with pH was reported for the oxidation
of testosterone by rat CYP2A1, CYP2B1, and CYP2C11 [14] and for
cyclosporine metabolism of human CYP3A4 [15]. Likewise, our re-
sults revealed a change in regioselectivity with pH and additionally
with buffer type. In light of previous literature on several P450 iso-
forms and substrates, these data suggest that ionic strength, as var-
ied by buffer concentration, may generally affect the activity of a
P450 reaction but not the regioselectivity, while pH may affect
both. However, further work with additional P450 isoforms and
substrates is needed to confirm this conclusion.
In conclusion, mass spectrometry was used to confirm the exis-
tence of multiple coupled catalytic modes for the CYP1A2 oxida-
tion of EOMCC in addition to the standard uncoupled modes. An
in situ optical method was developed to monitor the activity and
selectivity of human drug metabolizing enzymes and demon-
strated on a cyanocoumarin substrate. This method is anticipated
to be a useful tool for protein engineering studies seeking to under-
stand and control the influence of reaction conditions on P450
function. While the activity of CYP1A2 in this work was found to
vary over 10-fold as a function of buffer type, buffer concentration,
pH, and temperature, the regioselectivity and coupling efficiency
were effectively constant with buffer concentration and tempera-
ture but varied with buffer type and pH. Future work will investi-
gate the generality of these results with other P450 isoforms with
the aim of developing general heuristics relating P450 selectivity
and environmental parameters.
Acknowledgments
We thank Rita Nichiporuk and Ulla Anderson for assistance
with the mass spectrometry experiments and data interpretation,
and Jessica Ryan and Elizabeth Schneider for helpful discussions.
This work was supported by the National Science Foundation and
the National Institutes of Health.
It is relevant to emphasize that 7HCC is a minor product in the
overall oxidation of EOMCC by CYP1A2. This low regioselectivity
toward O-dealkylation suggests that studies employing 7-alkoxy-
3
-cyanocoumarin substrates as convenient fluorescent probes for
CYP1A2 activity are measuring only a fraction of the reaction prod-
ucts. A further elaboration of the product profile of commonly used
coumarin substrates would be a valuable contribution to the liter-
ature, especially in light of our current finding that 7HCC accounts
for only 21.7% of the substrate depletion.
Appendix A. Supplementary data
Because regioselectivity and coupling are governed by enzyme–
substrate binding interactions and neither temperature nor buffer
concentration altered regioselectivity or coupling, it follows that
these parameters do not alter the enzyme–substrate binding
interactions. Variations in P450 function with buffer concentration
have been previously attributed to the resulting variation in ionic
strength and not to specific effects of phosphate ions
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