The Mechanism of Laccases

The stoichiometry of the laccase catalyzed reaction (77, 42, 80) has long been established as  

With the exception of a study carried out with a partially characterized multicopper oxidase isolated from tea leaves (85), there has been very little detailed work concerned with the steady state kinetic behavior of laccases. Early work on the transient kinetics indicated, however, that (1) enzyme bound Cu + was reduced by substrate and reoxidized by O2, and (2) substrate was oxidized in one-electron steps to give an intermediate free radical in the case of the two electron donating substrates such as quinol and ascorbic acid. The evidence obtained suggested that free radicals decayed via a non-enzymatic disproportionation reaction rather than by a further reduction of the enzyme (86—88). In the case of substrates such as ferrocyanide only one electron can be donated to the enzyme from each substrate molecule. It was clear then that the enzjmie was acting to couple the one-electron oxidation of substrate to the four-electron reduction of oxygen via redox cycles involving Cu. 

The reduction potentials for the Cu sites were taken from Table 2 and the reduction potentials for the oxygen species from Ref. (92). 

In view of the energetic considerations a good deal of effort has been expended in determining the rates of reduction and oxidation of the Type 1 Cu center and the diamagnetic CuCu pair. The relevant observations for the two laccases are recounted below. 

---

Selinheimo, E., Autio, K., Kruus, K., Buchert, J. 2007. Elucidating the mechanism of laccase and tyrosinase in wheat bread making. JAgric Food Chem 55 6357-6365. 

T —type of copper o = oxidized, r—reduced Figure 61 The mechanism of laccase... 

The mechanism of laccase has been reported as a two-step reaction. The first step in the oxidation of quinol by laccase was the formation of the semiquinone, with the transfer of an electron from the substrate to the copper in the enzyme. The second step was a nonenzymatic disproportionation reaction between two semiquinone molecules to give one molecule of quinone and one of quinol. The function of copper and electron transfer in the reaction mechanism has been studied by using inorganic ions, electron paramagnetic resonance, and spectrophotometric methods [14]. 

While direct electron transfer to laccases may help elucidate the mechanism of action of these enzymes it is unlikely that this process will supply sufficient power for a viable implantable biocatalytic fuel cell, because of difficulties associated with the correct orientation of the laccase and the two-dimensional nature of the biocatalytic layer on the surface. However, a recent attempt to immobilize laccase in a carbon dispersion, to provide electrodes with correctly oriented laccase for direct electron transfer, and a higher density of electrode material shows promise [53],... 

Malmslrom, B. G. The mechanism of dioxygen reduction in cytochrome c oxidase and laccase. In Oxidases and Related Redox Systems (King, T. E., Mason, H. S., Morrison, M., eds.), Oxford-New York, Pergamon Press, 1981... 

The Mechanism of Oxygen Reduction in Oxidases—Studies with Laccase and Cytochrome Oxidase... 

The elaborate mechanism by which blue oxidases react with dioxygen to produce water was tackled by studying the possible role of H202. We have observed the formation of a stable and high affinity complex between tree laccase and H202. Moreover, the finding that the oxidation of the reduced enzyme with H202 follows a pattern which is different from that operative in the reduction of the oxidized enzyme may have important implications for the mechanism of action of laccase. 

The mechanism of dioxygen reduction at the trinuclear cluster in MCO catalysis has been a strong focus for research on this class of copper oxidases. Dioxygen reduction has been most thoroughly investigated in Rhus laccase (a plant laccase, from the Japanese lacquer tree). The primary reason for using Rhus Lac is the availability of a metal-substituted form the enzyme, a TlHg form, in which the... 

To better understand the mechanisms for the oxidation of lignin by a laccase mediator system, a laccase from Polyporous sp, kindly provided by Novozymes, was used in combination with 1-HBT. The redox mediator was found to be partly regenerated during the oxidation of lignin dimer 1 in the presence of laccase. A free radical of 1-HBT generated by laccase was probably responsible for the oxidation of I [146]. The free radical of 1-HBT was, however, transformed to benzotriazole, which could not mediate the oxidation of I. A proposed mechanism for the laccase mediator oxidation of nonphenolic lignins is given in Scheme 14.1. 

A Muheim, A Fiechter, PJ Harvey, HE Schoemaker. On the mechanism of oxidation of non-phenolic lignin model componnds by the laccase-abts conple. Holzforschung 46 121-126, 1992. 

The influence of [02], [laccase] on the performance of the laccase-initiated polymerization of vinyl monomers is essentially the same as observed with peroxidases (6.3.2) [69], Thus, the polymer characteristics are inversely correlated with [laccase] and [monomer], [Acac] exceeds only in minor effect on the polymer weight with the exception of very low [Acac] [69], Here, no polymerization was observed unless the [02] was reduced significantly. This indicates an 02-dependent irreversible inactivation mechanism of the Acac radical. The mechanism of this inactivation however remains to be elucidated. On the other hand, [02] may be an efficient handle to control the average molecular weight of the resulting... 

The kinetics if the anaerobic reduction of stellacyanin, plastocyanin, azurin, and laccase by [Fe(edta)] have been reported. Simple second-order behaviour was observed and the following rate constants, with their associated and values, were measured (at 25 °C and pH 7) 4.3x10 , 8.2x10 , 1.3x10 , and 2.6X 10 1 mol" s 3, 2, 2, and 13 kcal mol" and -21, -29, -37, and -5 cal K mol", respectively. The authors favour an outer-sphere mechanism for azurin, plastocyanin, and stellacyanin but conclude that laccase employs a pathway which requires specific protein activation (of ca. lOkcalmol" in A/f ) to accept the reductant. The kinetics of the reduction of laccase by [Fe(CN)6] are complicated, as they are for the autoxidation of reduced laccase. The results - for electron transfer between azurin and cytochrome c have already been mentioned. 

Observed total mass loss shows that non-ceUulosic components (pectin, hemicellulose, fats and waxes) are removed from fibre during enzymatic treatment, it has also been found that within the application of laccases followed by mechanical upgrading the mass loss is approximately twice as intensive as mechanical upgrading alone. The mass loss in a whole process is on the level of 20-21% for NS 51002 and 21-23% for NS 51003 while in mechanical processing it is only 11%. 

Laccase The role of laccase in the respiratory mechanisms Of the plant tissues still remains obscure. 

The authors reported the generation of polymers in the presence of a large excess of oxidant (monomer oxidant 1.6 1.0 vol/vol) without any information on the mechanism of polymerization, polymer molecular weight, or role and influence of oxidant on the enzyme or reaction kinetics. Subsequently, the laccase-catalyzed polymerization of acrylamide in water without any initiator (b-diketone) at temperatures ranging from 50 to 80 °C was reported however, the reactions were efficiently carried out at room temperature with the use of laccase/b-diketone (2,4-pentanedione) initiator to produce polyacrylamide in 97% yield (Mn = 2.3 x 10 ) [24,25]. The molecular weight of the polymer in these studies was determined by size exclusion chromatography. At 50, 65, and 80 °C when the polymerization was carried out for 4h, polyacrylamides with M = 9.4,9.2, and 10 x 10, respectively, were produced with the highest yield of 70% at 65 °C. 

Scheme 2.44 Plausible mechanism for the s5uithesis of quinoxaline derivatives in the presence of laccase at room temperature...

---