Leonard Pattenden wrote:
> [...] From my observations I would say there isn't any
> inhibitors to HIV-1 PR that would mimic the transition state,
Len,
your results are quite interesting. I would say that the interpretation
is somewhat complicated by the fact that nobody has ever /seen/ the
transision state for aspartic protease catalysis. Of course, people
often represent it as some kind of "tetrahedral intermediate", adding a
nucleophile to the peptidic carbonyl, but that's really just one part of
the structure, isn't it? How do we know what /other/ changes in the
substrate structure might occur simultaneously with the nucleophilic
attack?
> > I believe that co-operative effects ("subsite interactions") are largely
> > responsive for the failure of substrate vs. inhibitor correlations, but
> > perhaps even more importantly, /nonspecific/ interactions might be even
> > more important. We got our hands on one of those phenomena -- long
> > range, nonspecific, electrostatic interactions in pepsin catalysis
> > (http://www.biokin.com/papers/biblio.html#Kuzm9175). Those show
> > clearly that extrapolating from substrate kinetics (a /dynamic/ event)
> > to the binding of reversible inhibitors (a /static/ event) is very
> > tricky.
>> Interesting, I'll check it out.
Take a look. You will see a perfect straight line correlation between,
on the one hand, log(kcat/Km) for a series of peptide substrates and, on
the other hand, log(Ki) for a series of corresponding pseudopeptide
inhibitors. This is what Bartlett (and Wolfenden, ..., ...) used as
evidence that such inhibitors might be transition state analogs.
However, we have shown experimentally that the origin of this
correlation is in the /association rate constants/, k1 or k1* in the
scheme below:
k1 k2
E + S <====> ES ----> etc.
k-1
k1*
E + I <=====> EI* <====> EI
k-1*
You see, our substrates and inhibitors had a very high positive
electrical charge (several lysines and arginines at pH around 4), so
were able to modulate the association rate constant k1 (k1*) simply by
changind the /ionic strength/ of the buffer. Even though pepsin is an
approximately spherical macromolecule, as a first approximation we used
the Debye-Huckel theory to analyze the data and obtained an approximate
distance between the interacting ions (about 20 angstroms) as well as
the average surface charge of pepsin (isoelectric point about 1.5 !!)
that is "seen" by the charged ligands. Importantly, these /nonspecific/
interactions physically occur nowhere near the active site, and yet by
modulating the ionic strength (and thus k1, k1*) were able to change the
Km and kcat/Km (for substrates) or Ki (for inhibitors) by more than
three orders of magnitude! Importantly, the kcat stayed constant.
There was no "salting-in" effect because the overall ionic strength was
quite low.
> > Do you mean to say that HIV-Pr inhibitors toward which the virus becomes
> > resistant are /still/ potent in vitro inhibitors of the protease?? I
> > could just look up some recent papers from Jon Erickson's lab, but they
> > are not easily accessible for me right now.
>> That is exactly what I mean. Especially in my hands. It is impossible to
> predict selection via an assay system. I believe it is the nature of the
> assay or the protease, which co-embraces the ligand.
>> Unfortunately the assay I have inherited is apparently flawed It is at the
> wrong pH and salt concentration. This is significant as the pH optimum can
> vary by as much as 2 units depending on the particular substrate and can
> vary from 4-6, though it can operate from 3-7.5. All my assays are
> conducted at pH 6.5 to be more cytosol-like.I believe cytosolic pH is not
> relevant to HIV-1 PR activation and activity. The ionic strength has a
> great effect on the catalytic efficiency (Kcat/Km) and varies with ionic
> strength Km can change from milli--->micro in a transition from I= 0.1-2M,
> with Kcat increasing with I. Our assay uses I=0.1. However, the
> concentration inside a virion is likely to have a larger value of I and
> lower pH value.
Have you tried the fluorescence HIV-1 protease assay we developed? That
might fit your needs quite well. Here is the reference:
http://www.biokin.com/papers/biblio.html#Pera9542
Interestingly, the assay was developed by an /undergraduate/ student
(Ms. Anne Peranteau) working more or less independently with a gentle
nudging on my part. Nobody uses the assay except Jon Erickson's lab (as
far as I know). Funny: one might think that publishing a paper in
Analytical Biochemistry could cause at least some laboratories to try a
sensitive new assay. Wrong. I guess I forgot that science results seem
to travel most effectively by a word of mouth, and even then among
members of the same mutually supportive tribe. My favored quantum
chemistry textbook, by R. Zahradnik, has this motto printed on the book
jacket: "Everybody writes. Nobody reads." Seemed facetious at that
time, but not so much anymore. [;)]
-- Petr
_____________________________________________________________________
Petr Kuzmic, Ph.D. * BioKin, Ltd. * Consulting & Software Development
http://www.biokin.com * (608) 256-4790 * (608) 256-1269 FAX
