The rate of enzymatic reaction is proportional to the concentration of enzyme molecules. When the concentration of substrate molecules is high enough, the more enzyme molecules, the faster the substrate transformation rate. But in fact, when the enzyme concentration is high, this relationship is not maintained, and the curve gradually tends to be flat. According to the analysis, this may be caused by the high concentration of substrate with many inhibitors.
In biochemical reaction, if the concentration of enzyme is the fixed value and the initial concentration of substrate is low, the enzymatic reaction rate is proportional to the concentration of substrate, that means that it increases with the increase of substrate concentration. When all the enzymes combine with the substrate to produce intermediate products, even if the substrate concentration is increased, the intermediate product concentration will not increase and the enzymatic reaction rate will not increase. It can also be concluded that the enzymatic reaction rate is directly proportional to the initial concentration of enzyme under the same substrate concentration. The higher the initial concentration of the enzyme, the higher the enzymatic reaction rate.
In the actual determination, even if the enzyme concentration is high enough, the enzymatic reaction rate does not increase or even inhibited with the increase of substrate concentration. The reason is that high concentration of substrate reduces the effective concentration of water and the diffusivity of molecules, thus the rate of enzymatic reaction is reduced. Excess substrate gathers on enzyme molecules, resulting in inactive intermediate products, which can not release enzyme molecules, thus reducing the reaction rate.
In the optimum temperature range of various enzymes, the enzyme activity is the strongest and the enzymatic reaction rate is the highest. In the suitable temperature range, the enzymatic reaction rate can be increased by 1 ~ 2 times for every 10℃ increase in temperature.
The optimum temperature of enzymes in different organisms is different. For example, the optimum temperature of various enzymes in animal tissues is 37 ~ 40℃; The optimum temperature of various enzymes in microorganisms is 25 ~ 60℃, but there are exceptions, such as the optimum temperature of black koji glucoamylase is 62 ~ 64℃; The optimum temperature of glucose isomerase in Bacillus megaterium, Lactobacillus brevis, Aeromonas is 80℃. The optimum temperature of liquefied amylase of Bacillus subtilis is 85 ~ 94℃.
It can be seen that some Bacillus enzymes have high thermal stability. Too high or too low temperature will reduce the catalytic efficiency of enzyme, that means that the rate of enzymatic reaction is reduced.
For the enzymes which the optimum temperature is below 60℃, when the temperature reaches 60 ~ 80℃, most of the enzymes are destroyed and irreversibly denatured. When the temperature is close to 100℃, the catalytic effect of the enzyme is completely lost.
The enzyme shows activity in the optimum pH range, and the activity will be reduced if it is greater than or less than the optimum pH.
It is mainly manifested in two aspects:
①changing the charged state of substrate molecules and enzyme molecules, thus affecting the combination of enzyme and substrate;
②Too high or too low pH will affect the stability of the enzyme, and then the enzyme will suffer irreversible damage.
Substances that can activate enzymes are called enzyme activators.
There are many kinds of activators, mainly including:
①Inorganic cations, such as sodium ions, potassium ions, copper ions , calcium ions,etc.
②Inorganic anions, such as chloride ion, bromide ion, iodine ion, sulfate ion, phosphate ion, etc.
③Organic compounds, such as vitamin C, cysteine, reduced glutathione,etc.
Many enzymes show or strengthen their catalytic activity only when a suitable activator exists, which is called activation of enzymes. However, some enzymes are inactive after synthesis, and this enzyme is called zymogen. It must be activated by an appropriate activator before it becomes active.
Any substance that can reduce the activity of an enzyme without denaturing the protein of the enzyme is called an inhibitor of the enzyme. Factors that denature and inactivate the enzyme (called enzyme passivation), such as strong acid and alkali, are not inhibitors. It can reduce the rate of enzymatic reaction. Enzyme inhibitors include heavy metal ions, carbon monoxide, hydrogen sulfide, hydrocyanic acid, fluoride, acetic iodide, alkaloids, dyes, p-chloromercuric benzoic acid, diisopropyl fluorophosphate, ethylenediamine tetraacetic acid, surfactants and so on.
The inhibition of enzymatic reaction can be divided into competitive inhibition and non-competitive inhibition. The substance with similar structure to the substrate first binds to the active center of the enzyme, thus reducing the enzymatic reaction rate, which is called competitive inhibition.
Competitive inhibition is reversible inhibition, which can be finally released by increasing the substrate concentration and restore the activity of the enzyme. Substances with similar structures to substrates are called competitive inhibitors.
After the inhibitor binds to a site other than the enzyme active center, the substrate can still bind to the enzyme active center, but the enzyme does not show activity, which is called non-competitive inhibition.
Non-competitive inhibition is irreversible, and increasing substrate concentration can not relieve the inhibition of enzyme activity. An inhibitor that binds to a site other than the active center of an enzyme is called noncompetitive inhibition.
Some substances can act as both inhibitor of one enzyme and activator of another enzyme.