Laccase kinetic studies

Kinetic studies with laccase have shown that the enzyme must be reduced by the organic substrate before reaction with dioxygen occurs. The first electron from the substrate is accepted by the type 1 Cu2+, and the second by the type 2 Cu2+. The electrons from these reduced sites are then transferred to the type 3 copper pair, which then binds dioxygen with reduction to peroxide. It is possible that the type 2 and type 3 centres are in the same cavity, which only becomes accessible to the solvent when the type 1 Cu+ is oxidized. 

Faced with the problem of elucidating the individual roles of the diflFerent copper centers in the blue oxidases, the researcher has naturally focused in recent years on the laccases (9). Being easier to isolate, better characterized, and containing fewer copper atoms than cemloplasmin or ascorbate oxidase, the laccases from the Japanese lacquer tree Rhus vernicifera and the fungus Polyporus versicolor have been the subject of several transient kinetic studies in the millisecond range, that is, studies using stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy (9,49,50). 

MY Balakshin, CL Chen, JS Gratzl, AG Kirkman, H Jakob. Kinetic studies on oxidation of veratryl alcohol by laccase-mediator system. Part 1. Effects of mediator concentration. Holzforschung 54(2) 165-170, 2000. 

As previously mentioned, laccase is very closely related to ascorbate oxidase. The principal molecular architecture and arrangement of the mononuclear and trinuclear copper centers are the same. Furthermore, spectroscopic and kinetic properties are similar in many circumstances. Therefore, the catalytic mechanism of the dioxygen reduction should be the same for both. Kinetic studies on fungal and tree laccases have been... 

B. Reinhammar, Kinetic Studies on Polyporus and Tree Laccases. In Mutti-Copper Oxidases] A. Messerschmidt, Ed. World Scientific Publishing Singapore, 1997 pp 167-200. 

Liu, Z., Shao, M., Cai, R., Shen, P. 2006. Online kinetic studies on intermediates of laccase-catalyzed reaction in reversed micelle. J. Colloid Interface Sci. 294, 122-128. 

Figures 5.4 and 5.5 summarize results of a recent study of P. versicolor laccase electrochemistry based on cyclic and rotating disk voltammetry [60]. Figure 5.4 shows unequivocally that this laccase is voltammetrically active and gives a kinetically controlled, unpromoted four-electron peak at edge-plane pyrolytic graphite. Electrochemical reduction of 02 catalyzed by an immobilized laccase monolayer is close to reversible, and unrestricted by mass transport. The electrocatalysis follows, moreover, a Michaelis-Menten pattern (Fig. 5.5). Finally, there is a characteristic bell-shaped functional pH-profile with a pronounced maximum at pH 3.1.

In recent years kinetic and mechanistic studies have been done on the copper oxidases. Recent work has concentrated on laccase and tyrosinase, and since these two enzymes are good examples of the complexities involved, the rest of this paper concentrates on the enzymology of these two proteins. They also give rise to interesting comparisons since they have some substrates in common (e.g., catechol) but differ in certain aspects of their physicochemistry and mechanism. 

The catalytic cycle of multicopper oxidases is very complex because the four redox active Cu centers need to store reducing equivalents coming from four substrate molecules and transfer the electrons to dioxygen. From spectroscopic studies and fast kinetic experiments carried out on laccase it has been shown that reaction of fully reduced enzyme with O2 produces two intermediates, labeled as peroxy intermediate and native intermediate (Scheme 3). The peroxy intermediate has been trapped in a derivative of... 

Laccase, in contrast to GOase, does not mediate the aerobic oxidation of alcohols in vivo, but in the presence of a variety of electron mediators, notably TEMPO, is able to catalyze alcohol oxidations in vitro (47,50). Recent kinetic and EPR studies (58) have established the mechanistic details of TEMPO/laccase-catalyzed oxidations of alcohols and provided insights into reasons for the relatively low rates of conversion and the need for high loadings of TEMPO. This provides a sound basis for further improvement of the synthetic utility of this system, e.g., by stabilizing the laccase via immobilization and/or the use of more stable TEMPO derivatives. 

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 studies of the kinetics of bioelectrocatalytic transformations show that in some systems (for instance, adsorbed laccase ) the kinetic parameters correspond to the phenomenology of electrochemical kinetics, while in other systems (for instance, lactate oxidation they fit the phenomenology of enzymatic catalysis. In the latter case, we observe a hyperbolic dependence of anode current on the substrate concentration, as expected from the Michaelis-Menten equation. The absence of a general theory of bioelectrocatalysis does not permit us to examine the kinetics of electrochemical reactions in the presence of enzymes under different conditions. At present we can only try to estimate the scope of possible accelerations of electrochemical reactions by making some simple assumptions. 

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. 

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