Stability of Japanese-lacquer-tree (Rhus vernicifera) laccase to thermal and chemical denaturation: comparison with ascorbate oxidase.
The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located.
Agostinelli
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E
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Italy.
Cervoni
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L
Giartosio
A
A
Morpurgo
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L
D001204:Ascorbate Oxidase / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D042845:Laccase; D010088:Oxidoreductases / Q000737:chemistry*; D010944:Plants / Q000201:enzymology*; D011489:Protein Denaturation
D010088:Oxidoreductases; D042845:Laccase; D001204:Ascorbate Oxidase