Катехол оксидаза: структура, функция и механизм действия.

Enzyme

Catechol oxidase is a copper oxidase that contains a type 3 di copper cofactor and catalyzes the oxidation of ortho diphenols into ortho quinones coupled with the reduction of molecular oxygen to water. It is present in a variety of species of plants and fungi including Ipomoea batatas (sweet potato) and Camellia sinensis (Indian tea leaf). Metalloenzymes with type 3 copper centers are characterized by their ability to reversibly bind dioxygen at ambient conditions. In plants, catechol oxidase plays a key role in enzymatic browning by catalyzing the oxidation of catechol to o quinone in the presence of oxygen, which can rapidly polymerize to form the melanin that grants damaged fruits their dark brown coloration.

Polyphenol oxidases are a family of di copper metalloenzymes that include tyrosinase and catechol oxidase. In plants, both enzymes can catalyze the oxidation of ortho diphenols substrates into their corresponding ortho quinones. The key difference between the two related enzymes is that tyrosinase can catalyze the hydroxylation of monophenols to diphenols (monophenolase activity) as well as the oxidation of the o diphenol to the o quinone (diphenolase activity) whereas catechol oxidase only possesses diphenolase activity. When plant tissue is damaged, the chloroplast may rupture and release catechol oxidase into the plant cytoplasm, and vacuoles may also rupture, releasing stored catechol into the cytoplasm. The tissue damage also allows oxygen to penetrate into the cell. Thus, tissue damage facilitates the interaction of catechol oxidase with its substrate to produce o benzoquinone, which can polymerize non enzymatically to yield melanins that form an insoluble barrier for wound protection.

Catechol oxidase is nuclear encoded, and its N terminal end contains a signal peptide that directs the protein to the chloroplast thylakoid lumen, where it can either be soluble or loosely associated with the thylakoid membrane. Initially transcribed as a pro enzyme, the catechol oxidase precursor undergoes two rounds of proteolytic processing and transport before it enters the thylakoid lumen. Utilizing a [35S] methionine labeled precursor protein, Sommer et al. elucidated a proteolytic processing pathway common to a variety of plants including pea (Pisum sativum), tomato (Lycopersicon esculentum), and maize (Zea mays). The 67 kD precursor was imported into the stroma in an ATP dependent manner where a stromal peptidase processes the precursor into a 62 kD intermediate. The translocation of this intermediate into the thylakoid lumen was light dependent and results in the generation of the mature 59 kD enzyme. Based on analysis of the precursor and mature catechol oxidase purified from Ipomoea batatas, proteolytic processing removes both the N terminal transit peptide as well as a C terminal domain that covers the enzyme active site.

The crystal structure of catechol oxidase purified from Ipomoea batatas has been resolved in its active form in both the oxidized Cu(II) Cu(II) state and the reduced Cu(I) Cu(I) state. It is a globular, single domain monomeric enzyme that is approximately 55 by 45 by 45 Å in size and ellipsoid in shape. A four α helix bundle comprises the enzyme core, which girds the active site containing the dicopper center. The nitrogens on the imidazole side chains of His88, His109, and His118 coordinate with the first catalytic copper while the nitrogens on the imidazole side chains on His240, His244 and His274 coordinate with the second catalytic copper ion. In the oxidized Cu(II) Cu(II) state, each copper ion possesses a four coordinate trigonal pyramidal geometry, with the three histidine residues and a bridging hydroxide molecule forming the four ligands on each copper ion. Comparing the reduced (Cu(I) Cu(I)) state with the native (Cu(II) Cu(II)) state of the enzyme, the key difference is the distance between the two copper centers. In the oxidized Cu(II) Cu(II) state, the Cu Cu distance is 3.3 Å while in the reduced Cu(I) Cu(I) state, the distance increases to 4.4 Å. Furthermore, His109 bound to one of the copper centers is also covalently linked with Cys192 through a thioether bridge. This cysteine histidine cross linking may further restrain the enzyme active site from assuming the bidentate coordination complex readily formed in tyrosinase.

Although a crystal structure of catechol oxidase has been solved, questions concerning the exact mechanism of the reaction remain. One mechanism proposed by Eicken et al. is based on the crystal structure of catechol oxidase purified from Ipomoea batatas. This proposed catalytic cycle is supported by the experimental observation that stoichiometric amounts of o quinone form after catechol addition to the enzyme, even when dioxygen is absent. Other catalytic cycles elucidated with DFT calculations and crystal structures have been proposed which maintain the same charge in the active site throughout the cycle and thus do not require nearby bases. Enzymatic browning affects the nutritional quality and appearance of fruits and produce. Over half of fruit losses are estimated to occur as a result of enzymatic browning, and tropical produce are particularly vulnerable to this reaction. The key role of catechol oxidase in enzymatic browning makes it a common target for inhibition. While a number of inhibitory strategies exist such as high temperature treatments(70 90 °C) to eliminate catechol oxidase catalytic activity,

New approaches to design artificial enzymes based on amino acids or peptides as characteristic molecular moieties have led to a significant expansion of the field of artificial enzymes or enzyme mimics. Recent results by the group of Rob Liskamp have shown that scaffolded histidine residues can be used as mimics of certain metalloproteins and enzymes. The structural mimicry of certain copper proteins (e. g. hemocyanin, tyrosinase and catechol oxidase), containing type 3 copper binding sites, has been shown. This is a significant improvement since the use of scaffolded histidine residues is one step closer to the mimicry of enzymes by biologically relevant species.