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On a general note, it is not unusual at all for a random mutation (i.e., one not in the active or regulatory site of an enzyme, and not significantly connected with the catalytic or regulatory mechanism) to affect the rate constant of an enzyme-catalyzed reaction (kcat or kcat/Km) by a factor of 2 or more. Most enzyme kineticists don't get too excited about assigning an important role to a mutated residue unless one of the rate constants changes by a factor of 10. One danger in interpreting mutagenesis experiments is doing only a single kinetics measurement at a fixed pH and/or substrate concentration. If the mutation affects the pKa of enzyme catalytic groups or the effective Km of the substrate, a difference in rate will be noted, even though the fundamental rate constants kcat or kcat/Km, or their pH-independent maximal values, may not have significantly changed. Just my $0.02. -- ------------------------------------------------------------------------ Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] Mischa Machius wrote: > I assume you are talking about a sugar-binding enzyme ;) I have some > aspects to consider in addition to what Artem raises. Many effects of > a mutation are not recognizable in a static crystal structure or even > in an NMR structure. For example, it is usually difficult to assess > the thermodynamics of substrate binding, not to mention the kinetics. > Multi-valent substrates usually display some sort of cooperativity for > the binding process, which you might have affected by mutating one of > the subsites. You might be able to obtain some hints from a Michaelis- > Menten analysis of the mutant compared to the wild type, but that > would only be a start. Your crystallographic result of a less occupied > substrate-binding site for the mutant serves as a hint as well, but > such results are hardly conclusive. You will have to follow up with > more rigorous methods, such as ITC (thermodynamics of binding) and > time-resolved methods (kinetics of binding). > > One example of an effect of a mutation that is usually not > recognizable in a crystal structure has to do with substrate guiding. > In this case, the mutation has changed the surface of the protein, > thus affecting how well the multi-valent substrate can approach and > wiggle itself into the binding site. Once in the binding site, it is > structurally virtually indistinguishable from the wild-type. > > Ah, the nightmares of interpreting crystal structures in terms of > biology! > > Good luck! Best - MM > > > On Jun 11, 2008, at 7:21 PM, Narayanan Ramasubbu wrote: > > >> Dear all: >> I have a single residue mutant whose enzyme activity is about 50% of >> the wild type. Interestingly, the mutation >> is in a region that involves a secondary site but not the active >> site. The two structures with or without ligands >> fit well (0.18 A) and the metal binding and cofactor binding sites >> are all preserved in the mutant. The one difference >> noticed is that the ligand does not fill the active site (partially >> occupied subsites) unlike the wild type where all the >> subsites are occupied. Water structure around the actives site >> residues are "identical". >> >> I looked at the electrostatics and both surfaces look similar (not >> an expert). >> >> There are some residues whose sides chains show some positional >> disorder and these residues are at the edges of the >> active site. >> >> The resolution of the both data sets are 1.5A. >> The mutant enzyme was derived by MR. >> >> One another possibility that I want to look at is to compare the >> compactness of the two enzyme structures. >> What is the best way to compare that? I am wondering whether the >> "breathing" that was mentioned for some enzymes >> might be playing a role in the mutant enzyme. >> >> Also, I would appreciate comments on other possible explanations for >> this unusual (?) behavior. >> >> >> Thanks a lot >> >> Subbu >> > > > -------------------------------------------------------------------------------- > Mischa Machius, PhD > Associate Professor > Department of Biochemistry > UT Southwestern Medical Center at Dallas > 5323 Harry Hines Blvd.; ND10.214A > Dallas, TX 75390-8816; U.S.A. > Tel: +1 214 645 6381 > Fax: +1 214 645 6353 >