Ascorbate oxidase (ascorbic acid oxidase) is a copper-containing enzyme located in the cytoplasm or bound to the cell wall. Ascorbic acid oxidase is coupled with other redox reactions to function as a terminal oxidase, which catalyzes the oxidation of ascorbic acid, has an anti-aging effect, and plays an important role in the metabolism of substances in plants. Pyruvic acid, isocitric acid, α-ketoglutaric acid, malic acid, glucose-6-phosphate, and 6-phosphogluconate can all remove H protons by dehydrogenase and transfer H protons to coenzyme. Then, glutathione is used to transfer the H proton to ascorbic acid. Under the action of ascorbate oxidase, ascorbic acid is oxidized to O2, which combines with H protons to form water.
Ascorbate oxidase is a homodimeric enzyme with a molecular mass of 70 kDa and 552 amino acid residues per subunit (zucchini). The folding of all three domains is of a similar β-barrel type. The mononuclear copper site is located in domain 3 and the trinuclear copper species is bound between domains 1 and 3. A binding pocket for the reducing substrate that is complementary to an ascorbate molecule is located near the type-1 copper site and accessible from solvent. A broad channel providing access from solvent to the trinuclear copper species, which is the binding and reaction site for the dioxygen is present in ascorbate oxidase.
Enzyme Activity Assay
A commonly used method for determining ascorbate oxidase activity is the iodometric method, which is a classical method with a simple instrument and high accuracy. The second method is spectrophotometry, which has high sensitivity and accuracy, good reproducibility, wide application and good selectivity. The third method is the oxygen electrode analysis method, which has high sensitivity and good accuracy, but the oxygen electrode is very sensitive to temperature changes, and it is necessary to maintain a constant temperature during the measurement. The fourth method is pulse polarography, which is simple in operation and reproducible, consistent with the results of the iodometric method.
|Temperature||Ascorbic acid oxidase showed two peaks of activity at temperatures below 45 °C, and the enzyme activity lasted for a long time, up to 40 min, and the activity peak was advanced with increasing temperature. Ascorbate oxidase has a peak of enzyme activity at temperatures above 50 °C for a short duration, lasting only 20 min at 60 °C.|
|pH||Ascorbate oxidase exhibits higher activity in the pH range of 5-7, and the enzyme activity is the strongest at pH 5, which can reach 1.06 units. As the pH increases, the enzyme activity decreases. When the pH is 9, the enzyme activity is only 0.35 units.|
|Light intensity & nitrogen form||Under higher light intensity, the growth of plants supplying ammonium nitrogen was inhibited, the chlorophyll content of fresh leaf unit was significantly decreased, and the content of ascorbate oxidase was significantly higher than that of nitrate-nitrogen. In low light conditions, different nitrogen forms do not affect the ascorbate oxidase content.|
|Ozone||O3 can inhibit ascorbate oxidase activity, and the greater the concentration, the stronger the inhibition. O3 has the effect of maintaining ascorbic acid content.|
|Decompression||Decompression has a significant effect on inhibiting ascorbate oxidase activity, maintaining fruit firmness and ascorbic acid content.|
|Plant hormones||Among the common plant growth regulators, uniconazole and abscisic acid can increase the activity of ascorbate oxidase, and gibberellin can delay the increase of ascorbate oxidase concentration. 2,4-Dichlorophenoxyacetic acid can promote the activity of ascorbate oxidase at a certain low concentration.|
|Activator||Metal chelating agents (EDTA), reducing agents (NaHSO3), H3BO3, C6H8O7 and H2O are activators of ascorbate oxidase. This is due to the fact that these substances have different action characteristics against the ascorbate oxidase substrate or on the prosthetic group of the enzyme. Adding Cu2+ to the same enzyme solution has an inhibitory effect on ascorbate oxidase, which may be related to the destruction of ascorbic acid by metallic copper.|
|Metal ion||When plant tissues are treated with solutions containing different concentrations of Ca2+, Cu2+, Al3+, and K+, the concentration of ascorbate oxidase is greatly affected. Low concentration treatment has a tendency to inhibit enzyme activity, and as the concentration increases, the enzyme activity increases, and when a peak is reached, the activity of the enzyme is inhibited.|
Ascorbate oxidase is of great significance in plant physiology. Studies have shown that ascorbate oxidase activity is associated with wheat disease resistance and pepper resistance to aphids. After different treatments, some fruits such as kiwi, thorn pear, apple, and winter jujube can be kept fresh for a long time. If the metal ion stress conditions such as aluminum ion and calcium ion are used, the enzyme activity can be improved to some extent; in the presence of small organic molecules, the activity also changes to different degrees, which provides a reference for planting under different cultivation conditions.