RP1 Effect of a named variable on the rate of an enzyme-controlled reaction

To accurately measure how a specific concentration affects the rate, that concentration must actually be present.

Answer: Because as the reaction proceeds, substrate concentration decreases, becoming a limiting factor and slowing the rate.

Required practical 1. The initial rate is measured (usually by drawing a tangent at $t=0$) because at the very start of the reaction, the substrate concentration is known and is not yet acting as a limiting factor.

If investigating temperature (the independent variable), all other factors that affect enzyme activity must be controlled.

Answer: To maintain a constant pH, ensuring that any change in the rate of reaction is solely due to the temperature change.

Required practical 1. A buffer maintains a constant pH. pH is a control variable when investigating temperature; if the pH fluctuates, it could denature the enzyme, invalidating the results.

Boiling denatures the enzyme. If the reaction still happens, the enzyme wasn't doing the work.

Answer: To prove that the breakdown of the substrate is specifically catalysed by the active enzyme, and does not occur spontaneously.

A boiled enzyme control proves that the observed reaction relies on a functional enzyme with an intact active site, ruling out spontaneous breakdown or contamination.

If you mix a cold enzyme with a cold substrate and put them in a hot water bath, the initial reaction happens while the liquids are still cold.

Answer: To allow both solutions to equilibrate to the target temperature so the reaction occurs at the correct temperature from the very first second.

Required practical 1. Equilibration is crucial for validity. If the solutions are mixed before reaching the target temperature, the initial rate of reaction will be measured at a lower, incorrect temperature.

Rate is a measure of how fast something happens. If it takes a long time, the rate is low. If it takes a short time, the rate is high.

Answer: Rate = $1 / time$

Required practical 1. When the end-point of a reaction is measured by time taken (e.g., time for an 'X' to disappear in a milk powder/trypsin experiment), the rate is inversely proportional to time ($1/t$).

Buffers resist changes in hydrogen ion ($H^+$) concentration.

Answer: To keep the pH constant, ensuring the ionic bonds holding the enzyme's tertiary structure and active site together are not disrupted.

Required practical 1. pH affects the charges on the amino acids making up the active site. A buffer acts as a control variable, preventing pH fluctuations from acting as a confounding variable.

A negative control proves that the reaction will not happen *without* the independent variable (the active enzyme) present.

Answer: The substrate solution, the buffer, and a volume of distilled water equal to the volume of the enzyme solution.

Required practical 1. A negative control replaces the active component (enzyme) with an equal volume of a neutral substance (water) to prove that the breakdown of the substrate is entirely due to the enzyme and not spontaneous.

Enzymes are not used up, but the thing they are breaking down is.

Answer: The substrate concentration decreases over time and eventually runs out, meaning no more enzyme-substrate complexes can be formed.

Required practical 1. As the reaction proceeds, substrate is converted into product. The substrate concentration falls until it is fully depleted, at which point the reaction stops and the graph plateaus.