Leonard Pattenden wrote:
[...]
>> I'm in the "enzyme stretches the scissile amide bond" camp, but I don't
> think there will be a gross "flash" change at the TS. To go from the
> substrate to the transition state (if we assume the TS is accurately
> mimicked by the inhibitors) would take a lot of energy and would mean
> radical changes about the active site, but the whole thing looks much more
> subtle than that.
I agree that the structural changes during the HIV protease catalysis
(or in any enzyme, for that matter) are probably "much more subtle" than
many people originally assumed. In my thinking about these structural
changes, I was very influenced by reading Hans Frauenfelder's work on
myoglobin. Here is one of the most important references, which made a
huge difference in how I think about protein dynamics in general and
enzyme catalysis in particular:
FRAUENFELDER H, SLIGAR SG, WOLYNES PG
THE ENERGY LANDSCAPES AND MOTIONS OF PROTEINS
SCIENCE 254: (5038) 1598-1603 DEC 13 1991
More references follow at the end of my message. I haven't seen them
*all*, but I have seen most of them and they are eye-opening for any
enzymologist interested in structure / function relationships.
> Further, I have read in many papers that people believe the flaps close
> tighter on an inhibitor compared to a substrate.
Len, after you look closely at Frauenfelder's papers, you will probably
never stop asking yourself (as I do): what does it even mean to say "the
flap closes tigher", given that protein domains move constantly? This
discussion probably belongs to 'bionet.biophysics', but I will say here
at least that proteins are /dynamic/ molecules. According to
Fraunfelder's view of what he calls the "rugged energy landscape", any
structural change in an enzyme probably takes place via a very large
number of very small steps, or coordinated elementary motions, that
combine in a sequence, to produce the overall 'macroscopic' effect.
So we have here a common idea put forward by Athel a few days ago, but
now the same idea is presented in three different manifestations:
(1) it is rarely possible to assign a controlling or "rate limiting"
role to a single enzyme reaction in a metabolic sequence; it seems that
a better view is to see the entire cascade as a complete system, with
parts that are mutually interdependent in a complex fashion;
(2) on the next lower level in the molecular hierarchy, it seems rarely
even possible to ascribe some kind of "rate limiting" character to a
particular step in the given reaction mechanism (for the given enzyme);
(3) finally, descending even further onto the atomic level (talking
about molecular fragments, such as protein domains or aminoacid
side-chains), Frauenfelder et al. see in their experiments that protein
conformational changes /themselves/ are "composed" of very many tiny
motions, each of which represents only a very small barrier on the
potential energy surface ("rugged landscape", they call it). /Somehow/
on the system level the tiny energy changes get orchestrated into what
we see as global conformational change.
> From my structures I
> conclude that the interactions about the flap regions with the
> conserved water and ligand carbonyls, as well as the related H-bonding
> from G48/148 to ligand amides shows HIV-1 PR alters interactions and
> structure about the flap region and through the conserved water to satisfy
> all H-bonding interactions. This is particularly exemplified in the case
> of the inhibitor which binds very differently to both the substrate and
> product ligands, but still maintains good H-bonding through this region.
Following through with what I said above, I personally believe that our
attempts to see ligand binding mostly in terms of /separate/, highly
/localized/ interactions ("this H-bond", "this methyl group") are
somehow off the mark. In my opinion, it's the /totality/ of the very
many small interactions, in and /outside/ the enzyme active site, that
causes the substrate or enzyme to bind.
As far as binding /outside/ the active site followed by "sliding" of the
substrate (or inhibitor) into the catalytic site, it's very worthwile to
look up the work of Kuo-Chen Chou, who is now at Upjohn/Pharmacia [or
whatever conglomerate happens to own them now]. When you ask him, he
will happily send you re-prints of the work he's done at Shanghai prior
to his coming to the U.S. to work with Gordon Hammes. Chou has proposed
a kinetic theory of "nonspecific" intermolecular interactions that could
explain extremely high association rates for certain protein/DNA
interactions. The references to Chou's theoretical work (published in
the proceedings of the Chinese Academy during Mao's era!) are in these
papers of ours:
http://www.biokin.com/papers/biblio.html#Kuzm9175http://www.biokin.com/papers/biblio.html#Kuzm9119
> > 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.
>> Incredible! Perhaps when we think Ionic strength is modulating the enzyme
> in assays of HIV-1 PR by "salting in" the inhibitor it can also be
> modulating the enzyme directly by a non-specific interaction of surface
> residues quite analogous to pepsin? How very interesting.
It's very important to realize that we carefully constructed our test
system so as to /emphasize/ the ionic interactions, in a way that cannot
be reproduced in the HIV protease. Out of the total 330 or so
aminoacids, porcine pepsin has /many/ aspartates and glutamates
/uncompensated/ by basic aminoacids. Most of these acidic residues are
on the protein surface, which is the reason why at pH 4.0 pepsin
probably looks like one giant ball of negative charges. In contrast,
our substrates and inhibitors had a multitude of lysines and arginines,
so we have in fact engineered poly-ionic molecules with very high and
/opposite/ total charge. You can't do that with any other protease, or
most likely with any other enzyme for that matter. (Last time I looked,
pepsin has absolutely the lowest iso-electric point of all known
proteins.)
> I searched the
> structure for a long-range charge attractant like a carboxy-anion hole or
> something and found nothing except the highly conserved Arg 8/108, which
> is a whole other story in itself, I've made some compounds to "take
> out" these Arg's but it's a real can of worms. Perhaps the aspartyl
> proteases doesn't need a charge attractant? Perhaps, as for pepsin, if the
> pI is around 1.5 for the surface, perhaps this alone is enough to attract
> charged residues?
Exactly, as I explained above.
> The more I think about it the more I feel HIV-1 PR is subject to strict
> control. There is the cysteines which are subject to selective
> glutathionylation - one enhances, the other inactivates catalysis, there
> is the charged residues at p6 which act as an inhibitor at cytosolic pH,
> there is the preference for acidic pH and salt as in a budding virion,
Len, to be the devil's advocate, how many molecules of HIV protease do
you think migt be in the budding HIV virion? Just wondering if our
concept of pH and ionic strength is at all applicable to similar
biological objects. I think that enzyme kinetics in /membranes/,
/virions/, and organized (sequestered) structures like that would be
much better described using a completely different concepts, as well as
different mathematical formalism (e.g., Markov chains of statistics,
instead of the Mass-Action Law, the way neurologists think about the
kinetics of ion channels).
> I read a lot of papers, but only in the last six months has it become
> expedient to look at another assay. I also want to try some Microcal, like
> Todd et al have been pumping out. I am thinking of playing with Arg's to
> help bioavailability - something you've done by the sounds of it, did you
> try anything vs cells?
Nope. Plus I don't have a lab anymore (unless you want to invite me for
a visit, to see if I can still hold a pipettor), just writing computer
programs these days.
> I want to get my hands on the common drugs to try in my assay for direct
> comparison, do you happen to have any Saquinavir/ritonavir/Indinavir or
> VX-478 lying around?
I don't, but I can ask some doctor friends of mine, or maybe someone
else reading this can supply you with the goodies.
Best wishes,
- Petr
_____________________________________________________________________
Petr Kuzmic, Ph.D. * BioKin, Ltd. * Consulting & Software Development
http://www.biokin.com * (608) 256-4790 * (608) 256-1269 FAX
--------------------------------------------------------------
Selected bibliography of Hans Frauenfelder on protein dynamics
--------------------------------------------------------------
Frauenfelder H, Wolynes PG, Austin RH
Biological physics
REV MOD PHYS 71: S419-S430 Sp. Iss. SI MAR 1999
Frauenfelder H, Leeson DT
The energy landscape in non-biological and biological molecules
NAT STRUCT BIOL 5: (9) 757-759 SEP 1998
Frauenfelder H, McMahon B
Dynamics and function of proteins: The search for general concepts
P NATL ACAD SCI USA 95: (9) 4795-4797 APR 28 1998
Nienhaus GU, Muller JD, McMahon BH, et al.
Exploring the conformational energy landscape of proteins
PHYSICA D 107: (2-4) 297-311 SEP 1 1997
Frauenfelder H
Proteins: A challenging many-body problem
PHILOS MAG B 74: (5) 579-585 NOV 1996
Johnson JB, Lamb DC, Frauenfelder H, et al.
Ligand binding to heme proteins .6. Interconversion of taxonomic
substates in carbonmonoxymyoglobin
BIOPHYS J 71: (3) 1563-1573 SEP 1996
FRAUENFELDER H
COMPLEXITY IN PROTEINS
NAT STRUCT BIOL 2: (10) 821-823 OCT 1995
NIENHAUS GU, MOURANT JR, CHU K, et al.
LIGAND-BINDING TO HEME-PROTEINS - THE EFFECT OF LIGHT ON LIGAND-BINDING
IN MYOGLOBIN
BIOCHEMISTRY-US 33: (45) 13413-13430 NOV 15 1994
ABADAN Y, CHU K, CHIEN E, et al.
INFRARED STUDIES OF PROXIMAL-SIDE MUTANTS OF MYOGLOBIN
BIOPHYS J 66: (2) A360-A360 Part 2 FEB 1994
PARAK F, FRAUENFELDER H
PROTEIN DYNAMICS
PHYSICA A 201: (1-3) 332-345 DEC 1 1993
BRAUNSTEIN DP, CHU K, EGEBERG KD, et al.
LIGAND-BINDING TO HEME-PROTEINS .3. FTIR STUDIES OF HIS-E7 AND VAL-E11
MUTANTS OF CARBONMONOXYMYOGLOBIN
BIOPHYS J 65: (6) 2447-2454 DEC 1993
MOURANT JR, BRAUNSTEIN DP, CHU K, et al.
LIGAND-BINDING TO HEME-PROTEINS .2. TRANSITIONS IN THE HEME POCKET OF
MYOGLOBIN
BIOPHYS J 65: (4) 1496-1507 OCT 1993
PHILIPP RG, ERNST RM, NIENHAUS GU, et al.
LIGAND CONCENTRATION-DEPENDENCE OF THE REBINDING RATE OF CO TO MYOGLOBIN
BIOPHYS J 64: (2) A42-A42 Part 2 FEB 1993
NIENHAUS GU, MOURANT JR, FRAUENFELDER H
SPECTROSCOPIC EVIDENCE FOR CONFORMATIONAL RELAXATION IN MYOGLOBIN
P NATL ACAD SCI USA 89: (7) 2902-2906 APR 1 1992
STEINBACH PJ, CHU K, FRAUENFELDER H, et al.
DETERMINATION OF RATE DISTRIBUTIONS FROM KINETIC-EXPERIMENTS
BIOPHYS J 61: (1) 235-245 JAN 1992
ORMOS P, CHU K, EHRENSTEIN D, et al.
VIBRATIONAL STRUCTURE AND KINETIC HOLE-BURNING IN THE SORET BAND OF
SPERM WHALE MYOGLOBIN
FASEB J 6: (1) A54-A54 JAN 1 1992
MOURANT JR, NIENHAUS GU, FRAUENFELDER H, et al.
LIGHT-INDUCED CONFORMATIONAL TRANSITIONS IN SPERM WHALE MYOGLOBIN
FASEB J 6: (1) A54-A54 JAN 1 1992
YOUNG RD, FRAUENFELDER H, JOHNSON JB, et al.
TIME-DEPENDENCE AND TEMPERATURE-DEPENDENCE OF LARGE-SCALE CONFORMATIONAL
TRANSITIONS IN MYOGLOBIN
CHEM PHYS 158: (2-3) 315-327 DEC 15 1991
FRAUENFELDER H, SLIGAR SG, WOLYNES PG
THE ENERGY LANDSCAPES AND MOTIONS OF PROTEINS
SCIENCE 254: (5038) 1598-1603 DEC 13 1991
STEINBACH PJ, ANSARI A, BERENDZEN J, et al.
LIGAND-BINDING TO HEME-PROTEINS - CONNECTION BETWEEN DYNAMICS AND
FUNCTION
BIOCHEMISTRY-US 30: (16) 3988-4001 APR 23 1991
FRAUENFELDER H, NIENHAUS GU, JOHNSON JB
RATE-PROCESSES IN PROTEINS
BER BUNSEN PHYS CHEM 95: (3) 272-278 MAR 1991
HONG MK, BRAUNSTEIN D, COWEN BR, et al.
CONFORMATIONAL SUBSTATES AND MOTIONS IN MYOGLOBIN - EXTERNAL INFLUENCES
ON STRUCTURE AND DYNAMICS
BIOPHYS J 58: (2) 429-436 AUG 1990
BRAUNSTEIN D, ANSARI A, BERENDZEN J, et al.
LIGAND-BINDING TO SYNTHETIC MUTANT MYOGLOBIN (HIS-E7 GLY) - ROLE OF THE
DISTAL HISTIDINE
P NATL ACAD SCI USA 85: (22) 8497-8501 NOV 1988
ANSARI A, COWEN BR, FRAUENFELDER H, et al.
KINETIC HOLE-BURNING AND CONFORMATIONAL RELAXATION IN MYOGLOBIN
BIOPHYS J 53: (2) A274-A274 Part 2 FEB 1988
IBEN IET, BRAUNSTEIN D, FRAUENFELDER H, et al.
PROTEIN STATES AND MOTIONS PROBED BY PRESSURE-TEMPERATURE STUDIES OF CO
LIGANDS IN THE MYOGLOBIN POCKET
BIOPHYS J 53: (2) A275-A275 Part 2 FEB 1988
FRAUENFELDER H
FUNCTION AND DYNAMICS OF MYOGLOBIN
ANN NY ACAD SCI 504: 151-167 JUL 6 1987
ANSARI A, BERENDZEN J, BRAUNSTEIN D, et al.
REBINDING AND RELAXATION IN THE MYOGLOBIN POCKET
BIOPHYS CHEM 26: (2-3) 337-355 MAY 9 1987
