I am trying to find the best model to my data. The velocity (V) vs substrate [s] curve looks hyperbolic under normal conditions. When I introduce an inhibitor of interest at concentration of, say, 1 mM, the curve is low and flat. However, when the concentration of my inhibitor is at 0.5 mM, the curve is lower than the normal, but has a sigmoidal shape. This also occurs when the inhibitor under 1 mM is mixed in with one of its products of hydrolysis. Since the MM fit is not appropriate for all my curves I am looking at others, including the Hills equation because I believe the difference in shape has something to do with allosteric sites on my enzyme. I have a few questions:
1) I have been using the least squares method and Solver on Excel, with 'Vmax', 'Km' and 'n' as variable cells. These values are different for every curve, one results in n1. Am I solving correctly?
2) Am I using the right model and how do I know?
3) It appears that Vmax decreases and Km increases with increasing concentration of inhibitor. I can't figure out what inhibition this is.
Many thanks
Is this a single-substrate enzyme?
Is it possible that the inhibitor is an alternate substrate?
Have you checked for any kind of interference by the inhibitor with the readout?
Are you treating the inhibitor as non-competitive?
What do you know about the stoichiometry of inhibitor binding to the enzyme? Have you done any biophysical binding experiments?
Can you share the data?
Hi Adam, thanks for replying.
I am still trying to work out exactly what type of inhibition it could be.
What I know is that this enzyme has 2 hydroxyl-binding sites, 1 active site involving a serine, histidine and carboxyl triad, and 1 interfacial recognition site involving a carboxyl group. The catalytic mechanism involves an acyl-enzyme intermediate stabilised by hydrogen bonds.
I have not done any biophysical binding experiments myself, so I am not sure about the stoichiometry for this particular inhibiting compound.
Theoretically, there is a possibility that the inhibitor is also a substrate, as studies have shown this enzyme to hydrolyse the inhibitor. However, those studies were done in the absence of compounds required by the 2 hydroxyl-binding sites to change its conformation. Also, in the absence of the studied substrate, I have not observed any activity towards the inhibitor in any of my assays.
Elena
Hi Elena Venuti,
I know it has been a while since you had the problem you described above, I am facing the same problem at the moment...I exactly know how your curves must have looked like. Therefore I would be really interested if you somehow solved your inhibition-mystery ? Can you maybe share your findings? Thanks in advance
Melanie
Hi Melanie,
Sorry for the late response.
I managed to make some sense of it. Basically, it was a mixed, non-competitive type of inhibition.
I had 2 inhibitors, PC and LPC. They both are amphoteric surfactants with the same charge. The less potent of the two inhibitors, LPC, lacks one fatty acid chain which results in faster desorption from an emulsion interface in comparison with PC. I needed hyper-physiological quantities of LPC to reach the same level of inhibitoion of PC.
Mathematical modelling
The form of the Hill equation was:
v = Vmax [S]n/(Kbn+[S]n) (1)
where Vmax is the maximal velocity, [S] the substrate concentration, Kb the apparent dissociation binding constant, n the Hill coefficient (measure of enzyme cooperativity) and v the velocity (i.e., rate of reaction) at the given substrate concentration [S].
Parameters were obtained from measuring the velocity of the lipolysis by enzyme BSSL at different substrate concentrations, fitting the Hill equation. Hill coefficient was fixed at n = 2 during the fitting procedure.
The fitting was initially implemented in Microsoft Excel 2010 and, in a more refined form, in Wolfram Mathematica using the inbuilt ‘NonlinearModelFit’ routine. This delivered the best-fit values for kinetic parameters along with their standard errors.
Here is the link to my paper and supplementary tables of the regression model:
https://www.cysticfibrosisjournal.com/article/S1569-1993(17)30820-2/addons
I hope this helps.
Best,
Elena
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