I have queries related to Km, Kcat, Kcat/Km results. I have created point mutations in my proteins (beta-lactamases) and I have checked in vivo expressions. The mutant proteins seem inactive as indicated by MIC data. Purified mutant proteins too showed deviations from the non-mutant proteins in their kinetic parameters.
There are clear differences in Km values- mutants have higher km than non-mutant, subsequently decreased Kcat/Km ratio. But, there are minor differences in Kcat values. Although the Km values of mutants are higher yet, some have higher (2 fold) Kcat values than non-mutants. The overall Kcat/Km ratio is lower than non-mutants. What could be the possible explanation? Can we consider 2 fold Kcat hike as significant in mutants? Mutations may interfere both the Km values (affinity of the enzyme), Kcat values (turnover number) or may affect either.
Please provide your suggestions.
Thanks.
Provided that the affinity increases (lower Km) the reactions rate uses to decrease as the release of the product from the active site is more difficult. Of, course, this is not always so. There are some exceptions
I would consider a 2-fold difference in a kcat measurement between proteins to be within the range of experimental error. An increase in Km might be indicative of reduced substrate affinity, but this assumes that catalysis, rather than substrate binding, is rate limiting.
Hi Gaurav,
This is a 66-dollars question for sure ... there are NO definitive answers available unfortunately since 1) answers may change from one enzyme to another, 2) thorough kinetic invetsigations are needed to decipher what are the real impact of the mutation on the enzyme mechanism.
Adam is right in saying that a 2-fold difference is not "that" significant for steady-state kinetic parameters. I do agree too.
The catalytic efficiency paramater (kcat/KM ratio) is significant ONLY for a pure Michaelian enzyme (only in that case this ratio is directly proprotional to the transition energy of the enzyme). Most enzymes are not pure Michaelian and, thus, the kcat/KM ratio is at best a vague identification of what's going on in the active site.
So, what do we get? You have point mutations impacting mostly the KM value. As said by Adam, this means that affinity for the substrate is decreased in your mutants but not the kcat. That is a very interesting finding. Now position on the 3D structure of your enzyme the different mutations and deduce from the overall figure you get some possible explanations.
The question of KM being or not a good measure of the substrate affinity is a tricky one. The dissociation constant KD is a direct measure of the affinity of the substrate for the active site. KM may be quite different from KD, because KM is a macroscopic constant that is a combination, that can be very complex, of various microsocpic constants, including kon and koff that describe KD. However, KM is a geometrically determined constant: this is the concentration of substrate that corresponds to VMAX/2 on the hyperbolic representation. In that way, since it is a concentration of substrate affecting directly an observed phenomenon, KM is another measure of the substrate affinity and can be considered an accurate one to my opinion. Simple mathematics here.
Best regards,
Philippe
To add some more detail about Km versus Kd, as discussed by Philippe Urban, in terms of individual rate constants for the simplest case of a 1-substrate enzyme, according to Michaelis-Menten:
E + S ES E + P
Kd = k-1/k+1
Km = (k-1 + k2)/k+1
If k2 Kd.
For enzymes with more than one substrate, the expression for Km is still more complicated, but Kd for substrate n is still k-n/k+n
Thanks Philippe and Adam for your explanations. I am working on 3D modellings of the mutants to deduce a much better comprehension of the problem.
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