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> [email protected] <mailto:[email protected]> ---------- Group Leader, Adjunct Professor Faculty of Biochemistry and Molecular Medicine University of Oulu, Finland ---------- > On 13 Apr 2017, at 17:27, Pierre Nioche <[email protected]> > 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
