Enzymology

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.

Source

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.

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

Get My Free Ebook


Post a comment