A graph to show the effect of pH on an enzyme's activity:. Suggest an enzyme that would produce a trend as shown in the graph above. Pancreatic protease trypsin. Enzymes will work best if there is plenty of substrate available. As the concentration of the substrate increases, so does the enzyme activity. This means that more substrate can be broken down by the enzymes if there is more substrate available. This does not mean that the enzyme activity does not increase without end.
This is because the enzyme can't work any faster even though there is plenty of substrate available. So when the amount of available substrate exceeds the amount of enzymes, then no more substrate can be broken down. The enzyme concentration is the limiting factor slowing the reaction. As the concentration of the enzyme is increased, the enzyme activity also increases. This means that more substrate will be broken down if more enzyme is added.
Again, this increase in enzyme activity does not occur forever. So when the amount of available enzyme exceeds the amount of substrate then no more substrate can be broken down. The substrate concentration is the limiting factor slowing the reaction. Factors affecting enzyme action Physical factors affect enzyme activity. Temperature At low temperatures, the number of successful collisions between the enzyme and substrate is reduced because their molecular movement decreases.
How temperature affects enzyme action Higher temperatures disrupt the shape of the active site, which will reduce its activity, or prevent it from working. The substrates and enzymes were prepared in mM citrate-phosphate buffer at pH 6.
The enzymatic reaction was stopped every 10 min with the addition of mM sodium carbonate at pH The enzymatic activity at different time points of the [P] versus t curves was calculated based on the first-order derivative determined using the Origin 8. Deviations of the first derivative, i. Vertical dashed lines highlight three different assay times 20, 60 and min.
B Changes in the optimum temperature resulting from modifications of the enzyme assay duration. The enzyme concentration was nM. This complete dataset mean relative activities and respective deviations is presented on S1 Table. An overview of Fig 1 reveals that the relative position of the activity data associated with each temperature changes with assay time Fig 1A.
For example, at 20 min, the highest relative activity, i. Therefore, plots of the temperature effect on the relative enzyme activity for different assay durations clearly show different shapes and maxima, i. Therefore, the optimum temperature was also affected by the enzyme concentration. Inserts show the optimum temperature plot for min assays at each enzyme concentration.
Comparison of the inserted plots illustrates the optimum temperature shift due to enzyme dilution. This complete dataset mean relative activities and respective deviations is presented on S2 Table.
In conclusion, the optimum temperature changed with modification of the assay time and enzyme concentration. Thus, it is not a parameter that reflects an intrinsic enzyme property but is instead a mere consequence of the assay conditions. Briefly, at temperatures close to and above the enzyme melting temperature T m , the active enzyme concentration continuously decreases over the course of the assay due to thermal denaturation of the protein. The rate of protein denaturation is much lower below the T m , so the concentration of the active enzyme does not change in this temperature range.
Additionally, the higher the temperature, the larger the fraction of the substrate population that reaches the transition state, which increases the reaction rate.
These trends are simultaneous throughout the enzyme activity assay. Thus, at temperatures that allow the decrease in enzyme activity caused by protein denaturation to overcome the reaction rate increase caused by temperature, the detected enzyme activity drops during the course of the assay.
In contrast, at temperatures at which no protein denaturation occurs, the detected activity does not change as a function of time. For this reason, the relative activity data change during the assay, and the optimum temperature shifts toward lower values. Thus, bglTm is stable in the temperature range employed in the experiments, and consequently, the population of active enzyme does not change during the assay. The balance displacements as a function of assay temperature and enzyme T m do not apply here, and as expected, the relative activity data Fig 3A did not switch positions over time.
This plot did not depend on the assay time Fig 3B. A Relative activity of bglTm throughout the assay at different temperatures. B Effect of assay time on the relative activity. The enzyme concentration was 7. This complete dataset mean relative activities and respective deviations is presented on S3 Table. These remarks extend beyond a technical issue. Arrows indicate the purified proteins. The enzyme activity was determined based on the slope or the first derivative at each time point. More details are provided in the Material and Methods section.
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