I work with a thermophilic enzyme, its optimal temperature is 50 C. From some assays at 50 C and at 35 C, I was able to construct two lineweaver burke plots.
I want to ask you if my interpretation and understanding sounds correct. Comparing the Km/Vmax from either set, it appears that 35 C is a better temperature for activity. However, this enzyme experiences strong product inhibition and the higher temperature must be beneficial in reducing inhibition.
So....higher temperature = more energy = less binding efficiency = greater Km at 50 C ?
Since the product competes for binding to the active site, by reducing binding efficiency the exchange rate of product/substrate is improved and overall production is improved?
How would you report/identify the kinetic values for this enzyme? The true Km / Vmax would be at 50 C? How accurately should one describe "optimal" conditions before considering the Km / Vmax values for an enzyme? Would temp, pH and salt conc. be the main three conditions or are there others?
It sounds like you have conducted some experiments to compare the kinetic properties of an enzyme at two different temperatures, 50°C and 35°C, and have obtained Lineweaver-Burk plots for each condition. Here is a summary of what the data appears to be showing:
- At 50°C, the enzyme has a lower Km (lower affinity for the substrate) and higher Vmax (maximum rate of reaction) compared to 35°C. This suggests that the enzyme is more efficient at 50°C, in terms of its ability to catalyze the reaction.
- However, the higher temperature (50°C) may also be causing more product inhibition, which could be reducing the overall efficiency of the enzyme. Product inhibition occurs when the product of the enzyme-catalyzed reaction accumulates to a high enough concentration that it begins to bind to the enzyme's active site and inhibit its activity.
- The lower Km value at 50°C may be due to the fact that the higher temperature is providing more energy to the enzyme, allowing it to bind to the substrate more efficiently. However, this increased binding efficiency may also be contributing to the increased product inhibition at this temperature.
To accurately report the kinetic values for this enzyme, it would be important to consider not only the temperature but also the pH and salt concentration, as these can all have an effect on the enzyme's activity. Other factors to consider when describing "optimal" conditions for an enzyme include the presence of any required cofactors or activators, the presence of any inhibitors, and the concentration of the substrate.
In general, it's important to carefully consider all of the experimental conditions when interpreting enzyme kinetic data and to be mindful of any potential limitations or confounding factors that may be influencing the results.
lower Km = higher affinity
Also, I think you are stating the opposite. x and y intercepts are equal to (1 / Vmax) and (1 / Km), not Vmax and Km.
Hi there,
Have you actually checked the real pH value of your mix at each temperature? pH is varying with this parameter. And enzyme activity is also sensitive to pH variation.
Yes I have! This enzyme is a thermophilic and alkaliphilic, so I had to test a wide range of pH (5.5 - 11.5) and temperatures (25 - 65).
I had to adjust the pH of assay master-mixes for each temperature tested. Whew.
- The Lineweaver-Burk-plot, like all transformations of the dependent variable, suffers from a re-weighing of errors and thus high bias of the resulting regression parameters (since the independent variable is supposed to be error-free, its transformation has much smaller effect). Use a non-linear curve fitting procedure, preferably Nelder-Mead's simplex method (DOI:10.1093/comjnl/7.4.308 and its improvement https://www.cv.nrao.edu/adass/adassVI/kimys.html), and use bootstrapping to estimate the errors of the parameters (DOI:10.1016/0076-6879(92)10009-3).
- You have too few data points, for each curve you need at least 12 data points covering the range 0.1 to at least 5, better 10 times Km, which should be equidistant (DOI:10.1006/jtbi.1996.0023).
- If the data do not follow a simple Henri-Michaelis-Menten relationship (Lineweaver-Burk-plot is not linear), even more data points over a larger concentration range are required, in that case it makes sense to space the data points logarithmically and use lowest χ2 as fitting criterion, not lowest sum of squares (DOI:10.1046/j.1432-1327.1999.00643.x for an example). Discuss this with a statistician if required.
- For the plots of Km and Vmax against temperature, you have only 2 data points each. You need to measure a few more temperatures, especially as the relationship is unlikely to be linear.
- You can use the Vmax vs. temperature data to calculate the activation energy of the reaction (Arrhenius-plot) and the enthalpy and entropy from the affinity vs. temperature data (van't Hoff-plot).
While Dr. Buxbaum's remarks are all pertinent, I do not think that a more detailed analysis of the data (more data points, nonlinear fitting etc) could change the fact that your enzyme is more active at 35°C than at 50°C.
This is in contrast with your initial statement that "[the enzyme's] optimal temperature is 50°C".
Perhaps you meant to say that the organism from which the enzyme is derived grows optimally at 50°C?
If this were the case, and barring any mistakes in data collection (e.g., using two stocks of enzymes with different specific activities), there may be several reasons why your enzyme in vitro performs better at 35C than at the 'physiological' temperature of 50C.
I cite a few examples:
1) As you noted, the initial rates upon which your analysis is based have been collected in the absence of product - and, for that matter, of any inhibitor potentially existing in vivo. Physiological inhibitors may be binding more tightly at 35°C than at 50°C, thus offsetting the inherently greater efficiency of the enzyme at the lower temperature.
2) Conversely, if your assay mixture contains an inhibitor (e.g., some ion) that binds more tightly at 50°C, this could explain the in vitro pattern
3) pH 8.5 is a bith high, and may not reflect the in vivo pH at which the enzyme operates. Based on how temperature affects the pH-dependence of activity, testing the enzyme at, say, pH 7.5 might show a higher activity at the higher temperature.
4) Your isolated enzyme may be less stable at 50°C than at 35°C, wheras in vivo some interactor may stabilize its structure/acivity.
And so forth...
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