Yes, the pH optimum of a reaction can be changed with a mutation of one
of the catalytic residue or with a neighboring non-catalytic residue. A
classic example is the K166R mutation of mandelate racemase.
see: Biochemistry. 1995 Mar 7;34(9):2788-97
https://www.ncbi.nlm.nih.gov/pubmed/7893690
http://pubs.acs.org/doi/pdf/10.1021/bi00009a007
When lysine-166 is changed to arginine, one side of the reaction pH
profile shifts to the basic. If I remember correctly, the mutation
causes a 2 pH unit shift of the basic side of the pH reaction profile.
Steven Herron
sherron_...@yahoo.com
On 4/14/2017 1:35 PM, Petri Kursula wrote:
Hi,
apart from the possibilities of conformational flexibility affected by
crystal packing, just wondering if this mutation is supposed to
actually cause an increase or decrease in the corresponding activity
(outside the context of a crystal)? k(cat) and K(M) measured in
solution would help here. Is the pH in the crystal far from the
optimum of the wild-type protein? Can optimal pH change with the mutation?
Petri
Petri Kursula
----------
Professor
Department of Biomedicine
University of Bergen, Norway
http://www.uib.no/en/rg/petrikursula
<http://www.uib.no/en/persons/Petri.Kursula>
petri.kurs...@uib.no <mailto:petri.kurs...@biomed.uib.no>
----------
Group Leader, Adjunct Professor
Faculty of Biochemistry and Molecular Medicine
University of Oulu, Finland
----------
On 13 Apr 2017, at 17:27, Pierre Nioche
<pierre.nio...@parisdescartes.fr
<mailto:pierre.nio...@parisdescartes.fr>> wrote:
Dear CCP4bb,
We work on an enzyme that we crystallized with two substrates bound
in the active site (the reaction transform two substrates into two
products). We have also the structure with the two products. We are
able to see densities for the substrates when we collect data at
different time point post-crystallization (days or weeks later).
There is no change over time and no in crystallo enzymatic reaction
despite the fact that in solution using the same crystallization
solution, the reaction occurs readily.
This is not surprising and there are already many examples in the
literature.
However, when we crystallize a single amino acid variant (mutant
within the active site) with the same two substrates, we initially
see the substrates but we then observe in crystallo enzymatic
activity and formation of the final products over time. This
structure is identical to the one determined with the two products
co-crystallized with the enzyme. The crystal packing does not seem to
be at play here.
I understand that in crystallo activities are well documented in the
literature and can be induced by addition of ligands, X-rays, change
in oxidative environment, etc?
Here, the substrates are present from the beginning of the
crystallization experiments with the same concentration. Nothing is
added to the crystals later on. Only the time differentiate the two
type of crystals: after a couple of weeks, one has the substrates in
the active site (wt) while the other has the products (variant).
Is anyone aware of similar examples where a variant induce in
crystallo enzymatic activity without perturbation of the crystal?
Thanks,
Pierre
Dept of Pharmacology, Toxicology and cellular signaling
Paris Descartes University
