Laccase-like activity in the hemolymph of Venerupis philippinarum: characterization and kinetic properties.

The phenoloxidases (POs) include tyrosinases (EC 1.14.18.1), catecholases (EC 1.10.3.1) and laccases (EC 1.10.3.2) and are known to play a role in the immune defences of many invertebrates. For the Manila clam, Venerupis philippinarum, the exact role is not known, especially with regard to defences against Brown Ring Disease (BRD), which leads to high mortalities along European coasts. In order to understand the role and functioning of PO in V. philippinarum, the first step, and aim of this study, was to biochemically characterize the PO activity in the circulating hemolymph. Various substrates were tested and the common PO substrates L-DOPA, Catechol and dopamine exhibited good affinity with the enzyme and consequent low K(m) values (3.75, 1.97, 4.91 mM, respectively). A single tyrosinase-specific substrate, PHPPA, was oxidized, but the affinity for it was low (K(m) = 47.33 mM). Three tested laccase-specific substrates were oxidized by V. philippinarum PO (PPD, OPD and hydroquinone) and affinity was higher than for PHPPA. The results obtained with the substrate were confirmed by the use of different inhibitors: CTAB, a laccase-specific inhibitor inhibited PO activity greatly but not completely, whereas 4-Hr, specific to catecholases and tyrosinases, inhibited PO activity to a lesser extent. The results lead us to conclude that V. philippinarum PO activity in the circulating hemolymph, is mainly a laccase-like activity but there is also a smaller-scale tyrosinase-like activity. The inhibition mechanisms were also determined using dose-response substrate concentration for an inhibitor concentration equal or close to the IC50. Optimal conditions for the enzyme activity were also determined using L-DOPA as substrate, showing that its optimal temperature and pH are around 40 °C and 8.4 respectively. The enzyme is denatured for temperatures above 50 °C.