more P450 questions

Eli Hestermann ehestermann at whoi.edu
Thu Sep 12 10:22:55 EST 1996


Regarding "specific" substrates for P450s in general and 1A1 vs. 1A2 in
particular:

You are correct that EROD activity is performed by both 1A1 and 1A2.  If
the purpose of a particular experiment is to discriminate between the
two, then caffeine metabolism is a good choice.  This activity is
catalyzed by 1A2 but not 1A1.

This is a general problem with cytochromes P450 (CYP), in that catalytic
activity with regard to a single substrate may be performed by several
different CYP isoforms, i.e. there is considerable overlap among the
members of the superfamily in substrate specificity.  This is also a
concern with CYP knockout experiments.  Even though a specific enzyme is
knocked out, another member of the family may pick up the slack, so to
speak, and thus the knockout phenotype may not be as extreme as might be
expected given the known role(s) of the knocked out gene.  As you might
expect this also makes determination of the "true" endogenous role of
CYP isoforms difficult, as even though they may demonstrate catalytic
activity toward a substrate, it is difficult to demonstrate that that
particular activity is the endogenous role of that enzyme.

As for defining "specific" activity, that does seem to be a slippery
concept.  The case you mentioned seems to me to be a clear misuse of the
word, but I'm not sure a yes/no definition regarding catalytic activity
is the best approach either.  There are plenty of cases in the P450
literature where a purified enzyme demonstrates a measurable catalytic
rate with regard to a substrate, but that activity is far below what
most would consider physiologically important.  In such cases would it
not be better to first determine a level of in vivo activity which has
physiological relevance and then apply that standard when determining
whether a specific enzyme performs that activity?  Unfortunately that is
a much more difficult undertaking than simply reporting a measured rat
of activity.

I hope this helps.

Eli Hestermann

Eli Hestermann
ehestermann at mail.whoi.edu
Woods Hole Oceanographic Institution
Massachusetts Institute of Technology
"Vita brevis est, ars longa"    -Seneca

Chuck Miller wrote:
> 
> Hi Tox Fans,
> 
> While we're on the subject........
> 
> I have recently had cause to start reading the P450 literature in a
> critical manner. In many experiments the metabolism of a "specific"
> substrate is used to assess the presence and activity of a particular P450
> isoprotein. For instance, I have read papers stating that
> ethoyresorufin-O-deethylase (EROD) activity is specifically measuring
> CYP1A1 activity, while other investigators indicate CYP1A2 is also capable
> of metabolizing this substrate. Is or isn't ethoxyresorufin a "specific"
> substrate for CYP1A1? Is there a "specific" substrate that can discriminate
> CYP1A1 from 1A2 activity?
> 
> Perhaps the problem is that a more careful or better defined use of the
> word "specific" is needed, in that it implies that (as in this example
> above) all P450s (and other enzymatic activities as well) except the
> "specific one" have no activity against the substrate in question. This is
> the definition I have in mind when someone tells me they are using a
> "specific substrate" to assess a "specific" enzymatic activity. Is this a
> resonable/logical definition for "specificity" ?
> 
> Best of results,
> 
> Chuck
> 
> Dr. Charles A. Miller
> Dept. Environmental Health Sciences, SL29
> Room 374, Center for Bioenvironmental Research
> Tulane Univ. School of Public Health and Tropical Medicine
> 1430 Tulane Ave.
> New Orleans, LA 70112
> (504)585-6942



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