Is there any rules (should not be exact), to estimate the kinetic changes in an enzyme if I did any mutation on it?

If I cannot estimate the new kinetic values, is it possible at least to clarify or suggest any explanation for the new kinetic after mutating an enzyme depending on the point mutation I did on it?

So I want to explain the changes in the kinetic parameters (Km / Vmax) in my enzyme after mutating it, maybe depending on the 3D structure? or the charge/size of the new amino acids? Or any other factors I may use to suggest anything logical how and why those changes happened to my enzyme after mutating it?

I need that for my dissertation and I am still totally lost, any ideas (with the proper references) will be great. I really appreciate any help.

  • $\begingroup$ Any mutation affecting the interactions between the enzyme and the substrates, intermediates and products will change the enzymes behavior. I suggest reading some structural biology books and papers... Also please don't call it structural factors, that term is already used in crystallography. Finally you may want to add structural biology as a tag and possibly also ask the question at the chemistry stackexchange. $\endgroup$ Commented Oct 9, 2017 at 5:50
  • $\begingroup$ Computationally by molecular dynamics simulations coupled with free energy calculations (free energy perturbation or linear-approximation-based methods: linear response approximation and linear interaction energy. Visit PubMed and search for the relevant works by Arieh Warshel and Johan Aqvist. ncbi.nlm.nih.gov/pubmed $\endgroup$
    – user37894
    Commented Feb 15, 2018 at 18:10

2 Answers 2


Computationally, using molecular dynamics (MD) simulations, it is difficult but not impossible, to quantitatively reproduce the effect of mutations on ligand-binding free energies, and activation and reaction free energies of enzymatic reactions. The most useful MD simulation methods for such a purpose are: free energy perturbation (FEP), linear interaction energy (LIE), linear response approximation (LRA), empirical valence bond EVB), and their combinations---developed by Arieh Warshel (The Nobel Prize in Chemistry 2013).

Mechanistically, mutations affect short- (van der Waals and electrostatic) and long-range (electrostatic) interactions with the surrounding environment, which comprises the rest of the protein and the solvent. Note that free energy is an attribute of an ensemble, not of a single 3D structure---which is why exploring a single structure of the mutated protein is unlikely to reveal the effect of the mutation qualitatively, not to mention quantitatively. One has to sample the conformational space of the protein---and MD simulation is the most efficient computational technique for such a sampling.

In summary, the effects are simple, but the system is extremely complex . . .

Klvana et al. (2012) Biochemistry 51: 8829-8843.

Klvana et al. (2016) J. Phys. Chem. B 120: 13017-13030.


What you are asking is not completely clear: do you want to predict the effect of mutations? Or do you want to make sense of experimental kinetics data comparing the wild-type enzyme to point mutants?

To predict the effects of mutations, ideally you need a three-dimensional structure of the enzyme in complex with a substrate analogue. Is there such a structure in the PDB for your enzyme? This would help you reason about the potential effects of point mutations. If there is no such structure, you can only rely on sequence alignments (and possibly structures of homologous enzymes, if they have been solved) to figure out which residues are important for substrate binding and catalysis.

KM is related to the binding affinity of the enzyme for its substrate. So, any mutation that for example disrupts an H-bond between the enzyme and its substrate would increase KM.

Any mutation that targets a catalytic residue (say, an amino-acid side chain acting as an acid-base catalyst) would affect kcat (and therefore Vmax as well), and could make it larger or smaller depending on the mutation.

If you are trying to make sense of experimental kinetics data, then ask yourself why you chose these mutations in the first place. One usually mutates residues to test an hypothesis. Or are you trying to understand the effects of naturally occurring mutations?

  • $\begingroup$ do you want to predict the effect of mutations yes. Is there such a structure in the PDB yes. any mutation that for example disrupts an H-bond between the enzyme and its substrate would increase KM. why? that does not make sense for me. Without referencing any study which proves that I cannot rely on it. $\endgroup$ Commented Feb 15, 2018 at 4:41
  • 1
    $\begingroup$ Km is different than Kd (see textbooks for the precise definitions and meanings of both), but close enough so we can approximately consider Km as the binding affinity for the substrate. What causes binding? Well, any interaction between the enzyme and the substrate, including H-bonds, salt bridges, hydrophobic pocket on the enzyme that can accommodate the hydrophobic part of the substrate, etc. Disrupting any of these interactions will give a weaker binding affinity, therefore higher Kd and Km values. $\endgroup$
    – Guillaume
    Commented Feb 15, 2018 at 15:34
  • $\begingroup$ Regarding the available 3D structure of your enzyme: have you tried using it to figure out the potential effects of mutations? If so, how did you proceed, and what difficulties did you run into? Helping you would be easier if you told that. $\endgroup$
    – Guillaume
    Commented Feb 15, 2018 at 15:38
  • $\begingroup$ My problem is understanding how these factors you mentioned (H-bonds, hydrophobic pockets, etc.) may effect Vmax and Km differently? tighter binding (more bonds) between enzyme and substrate stabilizes the transition state, and that increases the Vmax, but I cannot get how Km will be impacted differently. In other words, I cannot distinguish which changes in the structure may change the Vmax, and which may change Km. $\endgroup$ Commented Feb 15, 2018 at 18:16
  • 1
    $\begingroup$ To the best of my knowledge (I am not an enzymologist), this is really hard to predict. Ultimately, the predictions you make from a structure need to be tested experimentally. The structure is a useful guide to narrow down the potential interesting mutations to only a few residues, because it clearly shows which ones have nothing to do with the catalytic site (i.e. most of them). $\endgroup$
    – Guillaume
    Commented Feb 15, 2018 at 20:39

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .