Testing the effect of enzyme concentration on enzyme activity requires a number of factors to be held constant. Understanding how enzymes function will make sense of why each factor needs to be fixed in place. The first factor that needs to be constant is the amount of substrate that is present. The second factor is the temperature of the reaction; it must be within the suitable range for the enzyme being tested. Third is the pH, or acidity, of the reaction. Each enzyme functions within a pH range of comfort. Last, there should be no enzyme inhibitors in the reaction mixture; otherwise the inhibitors will interfere with enzyme activity.

Keep It Coming

Enzymes operate on substrates, so without substrates for the enzyme to bind and modify there is no activity. If an enzyme runs out of substrates, it stops catalyzing reactions, so there will be no activity to measure. Increasing either the amount of enzyme or substrate will increase reaction rate. To test the effect of increasing enzyme concentrations on enzyme activity, the concentration of substrate must be held constant. This is accomplished by having a lot of substrate in the reaction mixture right from the start. An abundance of substrate during the time frame of the experiment ensures that the enzymes never run out of substrate, so there will always be activity that can be measured, even in test tubes that have a lot of enzyme. Having an abundance of substrate allows one to conclude that the different rates of activity in mixtures that contain different amounts of enzyme are due to the amount of enzyme, not that the substrate was all used up.

Nice and Warm

Enzymes are proteins that only function properly when they are in their proper three-dimensional shape. Protein structure is determined by intra-molecular interactions, or interactions between atoms within a protein. A protein maintains its proper three-dimensional shape only when these interactions are stable, holding the parts of a protein in place. Temperature, or the amount of heat, affects the intra-molecular interactions. Increasing the temperature of the reaction makes all atoms and molecules vibrate faster, which can speed up enzyme activity. Too high of a temperature will denature, or unfold, the enzyme, meaning there will be no more activity. Thus, it is important to hold the temperature of the reaction constant, and within the comfort zone of the enzyme, when testing the effect of enzyme concentration on enzyme activity.

The Litmus Test

Just as temperature can affect the three-dimensional shape of proteins, and thus their activity, the pH level of the reaction also affects enzyme shape and function. pH is the measure of how many protons -- positively charged hydrogen atoms -- are in a solution. The amount of protons can affect the intra-molecular interactions that hold proteins in shape. Each enzyme has an optimal pH at which it functions, and a comfort zone within which it will still function. Thus, it is important that the enzyme being studied be kept at the right pH and that the pH level does not change too drastically during the experiment.


Enzymes do not always work alone when they catalyze -- speed up -- a reaction. Sometimes enzymes need co-factors to help them bind to and modify substrates. Co-factors can be positively charged metal ions or complex molecules. Just as an enzyme's activity can be helped by another molecule, its function can be blocked, or inhibited, by other molecules. These inhibitors can compete with the substrate by binding to the enzyme’s active site, or they can change the shape of the enzyme by binding to it at places other than the active site. Thus, when testing the effect of enzyme concentration on enzyme activity, the amount of co-factors must be held constant and inhibitors should be absent.