It is believed that laccase catalysis involves (a) a reduction of T1 Cu by the reducing substrate, (b) an internal electron transfer from the T1 Cu to the T2/T3 trinuclear Cu cluster, and (c) a reduction of O2 to water at the T2/T3 Cu cluster [6, 11, 13]. The T1 and T3 Cu are linked mainly by a His-Cys-His tripeptide, whose Cys ligates the T1 Cu and whose His ligate two T3 Cu, and the T2/T3 Cu are electronically coupled to form a trinuclear cluster.

Having a confined access channel to and binding pocket at their T2/T3 Cu cluster, laccases strongly prefer O2 as their oxidizing substrate. Having a much more open and shallow pocket at their T1 Cu site, however, laccases have a low specificity towards their reducing substrates [17-22]. Substrates whose redox site resides on freely rotatable benzene, benzothiazoline, or moieties of similar dimensions may easily dock inside the T1 pocket. A wide range of redox-active metal complexes, anilines, thiols, and especially phenols can transfer electrons to laccases, given that their E° is -1 V or less. A KM in the order of 0.1 mmol L-1

and a kcat in the order of 103 s 1 are often observed for a typical reducing substrate, and a Km in the order of 0.05 mmol L-1 and a kcat in the order of 102 s-1 are often observed for O2 [1, 8, 28].

For many reducing substrates, their reactivity tends to correlate with the difference between their E° and that of laccases' T1 Cu, suggesting an "outer sphere" type of electron-transfer mechanism in which the activation energy is regulated mainly by the thermodynamic driving force, the E° difference (AE °) [29-34]. Compared with a low-E° counterpart, a high-E° laccase may not only possess a higher oxidation potency (to work on more recalcitrant substrates) but also oxidize a substrate faster, making such an enzyme more attractive as an industrial catalyst.

Laccases are often able to oxidize substrates with an E ° exceeding that of their T1 Cu, because the apparent endothermic oxidation half-reaction may be compensated by the vastly exothermic O2 to H2O reduction half-reaction, yielding an overall negative Gibbs' free energy change. However, such energetics would diminish at alkaline pH, when the pH-sensitive E° of O2/H2O is lowered close to or below that of laccases. For instance, the E° of O2/H2O is ~1.0, 0.8, and 0.6 V at pH 4, 7, and 10, respectively. In the range of pH 4-10, the E° (T1) of Trametes vil-losa and Myceliophthora thermophila laccase is about 0.8 and 0.5 V, respectively [32]. Thus, thermodynamically, T. villosa laccase would become inactive above pH ~7 to oxidize a substrate with an E° > 0.8 V, and M. thermophila laccase would become inactive above pH ~11 to oxidize a substrate with an E° > 0.5 V. Sometimes, an initial endothermic electron transfer from a high E° substrate to laccase may also be compensated by coupled chemical reactions (for example, deproton-ation of an N—OH cation radical) [35].

In general, a bell-shaped pH-activity profile (with optimal pH (pHopt) at —5—7) is observed for phenols, anilines, or other substrates whose oxidation by laccases is accompanied by H+ dissociation. Because of the oxidative H+ release, the E° of these substrates decreases as pH increases. The subsequent increase of the AE ° with laccase enhances the enzymatic oxidation, contributing to the ascending part of the pH profile. At higher pH, however, the laccase inhibition by OH-becomes more pronounced, contributing to the eventual descent of the pH profile [29, 32, 36]. For 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hexacyanidoferrate(4), or other substrates whose oxidation by laccase is not accompanied by H+ dissociation, a monotonic pH-activity profile is often observed within pH 4—9, attributable to the relative insensitivity of their E ° to pH. Proton-ation—deprotonation of the substrates and/or laccase might also affect the pH profile.

Most fungal laccases are mesophilic, with optimal temperature (Topt) at —60 °C. These laccases could quickly be inactivated at temperature above —50—60 °C [1—3, 5—10, 12]. The thermal instability might be caused by protein unfolding or Cu loss. However, a laccase from the thermophilic Chaetomium thermophilum has a Topt of —70 °C [37].

Laccase can be inhibited by various reagents, including halides, sulfanyl groups, and cationic quaternary ammonium surfactants [8]. Small "hard" anions such as

Table 2.2 General enzymological properties of typical laccases.


Km (mmol L-1) kc„ (s-1) KM (O2) (mmolL-1) pHopt Topt (°C)

Bacterial ~10-1-10°

60 80

F-, OH-, CN-, and N- can tightly bind to the T2 Cu, interrupting the internal electron transfer and/or O2 activation. Thiols and sulfur-liking metal ions such as Hg2+ can cleave the T1 Cu-Cys ligation. Reductants may also leach Cu out from laccases.

Table 2.2 summarizes some of the enzymological properties of laccase often relevant to its applications.

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